Large scale enzymatic synthesis of oligosaccharides

ABSTRACT

A novel UDP-Gal regeneration process and its combined use with a galactosyltransferase to add galactose to a suitable acceptor substrate. Also described herein are synthetic methods for generating Globo-series oligosaccharides in large scale, wherein the methods may involve the combination of a glycosyltransferase reaction and a nucleotide sugar regeneration process.

RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Patent Application No. 61/684,974, filed Aug. 20, 2012,which is herein incorporated by reference in its entirety.

BACKGROUND

Globopentaose (Gb5), fucosyl-Gb5 (Globo H), and sialyl-Gb5 (SSEA4) areglobo-series glycosphingolipid and were first discovered in 1983 incultured human teratocarcinoma cell line^([1]) and subsequently found inseveral malignant cancers.^([2],[3]) Report showed Globo Hoverexpression in up to 61%, Gb5 overexpression in 77.5% and SSEA4overpression in 95% in breast cancer patients.^([4]) On the other hand,HER2 gene, the target for therapeutic monoclonal antibody Trastuzumab(Herceptin) that interferes with the HER2/neu receptor, is overexpressedin only 25% breast cancer patients^([5]). The comparison clearlydemonstrated that the glycosphingolipid antigens (Gb5 and itsderivative, Globo H and SSEA4) are better candidates to be developedinto cancer vaccine. Hence, Globo H has been conjugated to the keyholelimpet hemocyanin (KLH) as a cancer vaccine, and is under phase IIclinical trial in some country now.^([6])

There are several disadvantages of current methods used for thesynthesis of Gb5, Globo H and SSEA4. The traditional chemical synthesisis tedious and labor-consuming, and several protection and de-protectionsteps are necessary to achieve high purity and correct stereotype andalways lead to the very low total yields. Till now there are manyreports for the chemical synthesis of GloboH.^([7] [8] [9] [10] [11] [12] [13] [14]) However, only two reports havebeen published for SSEA4 synthesis. Hsu et al reported a one-potchemical synthesis approach to assembled the glycan part of SSEA-4 in24% yield^([15]) Zhen et al. reported the use of a chemoenzymatic methodto synthesize SSEA-4 in milligram scale.^([16]) On the other hand, theenzymatic synthesis of Globo H based on Leloir-type glycosyltransferaseonly requires the active nucleotide sugar as donor to catalyze theglycosylation reaction. Nonetheless, the nucleotide sugar is tooexpensive to synthesize in large scale. Moreover, the by-productpyrophosphate and nucleoside diphosphate inhibit the nucleotide sugarformation of pyrophosphorylase^([15]) and Leloir-typeglycosyltransferase; therefore, how to develop a regeneration strategyis necessary to overcome the limitation and to recharge the nucleotideto achieve constitute nucleotide sugar product in order to continue thereaction. During the past several years, many groups worked to tacklethe major problem of nucleotide sugar regeneration and most of the sugarnucleotide regeneration have been solved. However, there is still somespace to improve the technology of sugar nucleotide regeneration,especially the UDP-Gal regenerate is much difficult. For example,UDP-Gal regeneration was first proposed in 1982 by Wong and Whitesidevia UDP-Glc C4 epimerase to interconverse UDP-Glc and UDP-Gal (^([17])).Ten years later, our group developed the secondary UDP-Gal regenerationmethod. Instead of using UDP-Glc C4 epimerase, Glc-1-phosphateuridylyltransferase located in galactose operon in E. coli was used tointerchange Gal-1-phosphate and UDP-Glc to Glc-1-phosphate andUDP-Gal.^([18]) However, the final pathway to directly condense UTP andGal-1-phosphate to form UDP-Gal was not established due to the absenceof suitable enzyme. Because the target compounds Gb5, Globo H and SSEA4ae Gal-related molecules, how to overcome the major difficult of UDP-Galregeneration and increase its efficiency will be the key point for largescale enzymatic synthesis of Gb5, Globo H and SSEA4.

In summary, there are several limitations to current methods of largescale synthesizing Gb5, Globo H and SSEA4 in the art. Thus, there is aneed for new synthetic procedures that produce Gb5, Globo H, SSEA4, andintermediates thereto in an efficient manner.

SUMMARY OF THE INVENTION

The present disclosure is based on the development of new nucleotidesugar regeneration processes and their applications in sugar synthesis.Such sugar synthesis methods, involving the combination of at least onenucleotide sugar regeneration system (e.g., the UDP-Gal regenerationsystem described herein) and at least one glycosyltransferase (e.g.,galactosyltransferase), were used in synthesizing variousoligosaccharides (tailed), including allyl-tailed Gb3, Gb4, Gb5 (alsoknown as SSEA3), Fucosyl-Gb5 (also known as Globo H), and Sialyl-Gb5(also known as SSEA4), with unexpectedly high efficiency and yields.More specifically, the synthetic approaches described hereinunexpectedly allow chain reactions to produce final products, such asGlobo H and SSEA4, without the need to purify intermediates.

Accordingly, one aspect of the present disclosure relates to methods foradding a galactose residue to a substrate via the action of agalactosyltransferase coupled with a UDP-Gal regeneration process. Themethod comprises: (i) producing UDP-Gal from galactose in the presenceof a set of UDP-Gal regeneration enzymes, wherein the set of UDP-Galregeneration enzymes comprises a galactokinase, an UDP-sugarpyrophosphorylase, a pyruvate kinase, and optionally, a pyrophosphatase;(ii) reacting the UDP-Gal with a substrate molecule (e.g., apolysaccharide, an oligosaccharide, a glycoprotein, a glycolipid, or anaglycone) via action of a galactosyltransferase (e.g., analpha1,4-galactosyltransferase, a beta1,4-galactosyltransferase, analpha1,3-galactosyltransferase, or a beta1,3-galactosyltransferase) toadd a galactose residue to the substrate molecule; and, optionally,(iii) isolating the galactosylated product thus produced. Steps (i) and(ii) can take place in a reaction mixture comprising the set of UDP-Galregeneration enzymes, the galactosyltransferase, the substrate molecule,galactose, ATP, and UTP. In some examples, the substrate molecule is aceramide or a glycosphingolipid.

Another aspect of the present disclosure relates to methods forsynthesizing oligosaccharides involving at least one nucleotide sugarregeneration process (e.g., UDP-Gal regeneration) and at least onereaction of adding a monosaccaride, e.g., galactose (Gal),N-acetylgalatocoamine (GalNAc), fucose (Fuc), and sialic acid (Neu5Ac),onto a suitable acceptor via action of a glycosyltransferase, e.g.,galactosyltransferase, fucosyltransferase, sialyltransferase, andN-acetylgalactosaminyltransferase.

In some embodiments, the method described herein for enzymaticallysynthesizing an oligosaccharide, uses lactose (e.g., tailed) as thestarting material. The method comprises: (i) producing UDP-Gal fromgalactose in the presence of a set of UDP-Gal regeneration enzymes,wherein the set of UDP-Gal regeneration enzymes comprises agalactokinase (e.g., from E. coli), an UDP-sugar pyrophosphorylase(e.g., from A. thaliana), a pyruvate kinase (e.g., from E. coli), andoptionally, a pyrophosphatase (e.g., from E. coli); (ii) convertingLac-OR^(1A) into Gb3-OR^(1A) in the presence of the UDP-Gal and analpha-1,4 galactosyltransferase (e.g., a LgtC such as that from N.meningitides), wherein R^(1A) is hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted alkenyl, substituted orunsubstituted alkynyl, substituted or unsubstituted carbocyclyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, or an oxygen protectinggroup. Lac-OR^(1A) refers to lactose(β-D-galactopyranosyl-(1→4)-D-glucose) (e.g., also encompassed byFormula (I), wherein each of R^(2A), R^(3A), R^(5A), R^(2B), R^(3B), andR^(5B) is hydrogen) wherein the group attached to the anomeric carbon oflactose is an —OR^(1A) group, and wherein R^(1A) is as defined herein.

Examples of R^(1A) include, but are not limited to hydrogen, allyl,biotin, a ceramide, or a non-hydrogen group (e.g., alkyl) which isfurther substituted with a substituted or unsubstituted thio,substituted or unsubstituted amino, carbonyl (e.g., carboxylic acid),azido, alkenyl (e.g., allyl), alkynyl (e.g., propargyl), biotin, or aceramide group. In certain embodiments, R^(1A) is hydrogen, allyl,substituted alkyl, biotin, or a ceramide.

When necessary, Gb3-OR^(1A) can be isolated from the reaction mixture.

Steps (i) and (ii) can occur in a Gb3-synthesis reaction mixturecomprising galactose, PEP, ATP, UTP, the Lac-OR^(1A), thealpha-1,4-galactosyltransferase, and the set of UDP-Gal regenerationenzymes. In one example, the molar ratio of the Lac-OR^(1A) andgalactose in the Gb3-synthesis reaction mixture is 1:1 before occurrenceof any enzymatic reactions.

Any of the methods described above can further comprise: (iii)converting the Gb3-OR^(1A) into Gb4-OR^(1A) in the presence ofUDP-GalNAc and a beta1,3-N-acetylgalactosaminyltransferase (e.g., a LgtDfrom a suitable organism such as H. influenza), which can be coupledwith (iv) producing the UDP-GalNAc from GalNAc in the presence of a setof UDP-GalNAc regeneration enzymes, wherein the set of UDP-GalNAcregeneration enzymes comprises an N-acetylhexosamine 1-kinase (e.g.,from B. longum), an N-acetylglucosamine 1-phosphate uridyltransferase(e.g., from E. coli), and a pyruvate kinase (e.g., from E. coli), andoptionally, a pyrophosphatase (e.g., from E. coli). Steps (iii) and (iv)can be carried out in a Gb4-synthesis reaction mixture comprisingGalNAc, PEP, ATP, UTP, the Gb3-OR^(1A), thebeta1,3-N-acetylgalactosaminyltransferase, and the set of UDP-GalNAcregeneration enzymes. In one example, the Gb4-synthesis reaction mixtureis prepared by mixing the Gb3-synthesis reaction mixture with at leastGalNAc, the beta1,3-N-acetylgalactosaminyltransferase, theN-acetylhexosamine 1-kinase, and the N-acetylglucosamine 1-phosphateuridyltransferase. When necessary, Gb4-OR^(1A) can be isolated from thereaction mixture.

After synthesis of Gb4-OR^(1A), the method as described above canfurther comprise: (v) converting the Gb4-OR^(1A) into Gb5-OR^(1A) in thepresence of UDP-Gal and a beta1,3-galactosyltransferase (e.g., a LgtDsuch as that from H. influenza), which can be coupled with (vi)producing the UDP-Gal from galactose in the presence of the set ofUDP-Gal regeneration enzymes described herein. In one example, (v) and(vi) take place in a Gb5-synthesis reaction mixture comprisinggalactose, PEP, ATP, UTP, the Gb4-OR^(1A), thebeta1,3-galactosyltransferase, and the set of UDP-Gal regenerationenzymes. The resultant Gb5-OR^(1A) can be isolated from the reactionmixture.

The above method can further comprise steps for converting theGb5-OR^(1A) thus obtained into Fucosyl-Gb5-OR^(1A) (Globo H) or intoSialyl-Gb5-OR^(1A) (SSEA4).

For Globo H synthesis, the method can further comprise: (vii) convertingthe Gb5-OR^(1A) into Fucosyl-Gb5-OR^(1A) in the presence of GDP-Fuc andan alpha1,2-fucosyltransferase (e.g., from H. pylori), which can becoupled with (viii) producing the GDP-Fuc from fucose in the presence ofa set of GDP-Fuc regeneration enzymes, wherein the set of GDP-Fucregeneration enzymes comprises a L-fucokinase/GDP-fucosepyrophosphorylase (e.g., B. fragilis), a pyruvate kinase (e.g., from E.coli), and a pyrophosphatase (e.g., from E. coli). In one example, steps(vii) and (viii) occur in a Fucosyl-Gb5-synthesis reaction mixturecomprising fucose, ATP, GTP, PEP, the Gb5-OR, thealpha1,2-fucosyltransferase, and the set of GDP-Fuc regenerationenzymes. The Fucosyl-Gb5-synthesis reaction mixture can be prepared bymixing the Gb5-synthesis reaction mixture with at least fucose, GTP, thealpha1,2-fucosyltransferase, and the L-fucokinase/GDP-fucosepyrophosphorylase. When necessary, the resultant Fucosyl-Gb5-OR^(1A) canbe isolated from the reaction mixture.

For SSEA4 synthesis, the method can further comprise: (ix) convertingthe Gb5-OR^(1A) into Sialyl-Gb5-OR^(1A) in the presence of CMP-Neu5Acand an alpha2,3-sialyltransferase (e.g., from M. bacteria), and (x)producing the CMP-Neu5Ac from Neu5Ac in the presence of a set ofCMP-Neu5Ac regeneration enzymes, wherein the set of CMP-Neu5Acregeneration enzymes comprises a cytidine monophosphate kinase (e.g.,from E. coli), a CMP-sialic acid synthetase (e.g., from P. Multocida), apyruvate kinase (e.g., from E. coli), and optionally a pyrophosphatase(e.g., from E. coli). Steps (ix) and (x) can occur in aSialyl-Gb5-synthesis reaction mixture comprising Neu5Ac, CTP, PEP, theGb5-OR^(1A), the alpha 2,3-sialyltransferase, and the set of CMP-Neu5Acregeneration enzymes. The Sialyl-Gb5-synthesis reaction mixture isprepared by mixing the Gb5-synthesis reaction mixture with at leastNeu5Ac, CTP, the alpha 2,3-sialyltransferase, the cytidine monophosphatekinase, and the CMP-sialic acid synthetase. The Sialyl-Gb5-OR^(1A) canthen be isolated from the reaction mixture.

In one example, a method for synthesizing Globo H can be performed asfollows: (i) producing UDP-Gal from galactose in the presence of theUDP-Gal regeneration enzymes as described herein, (ii) convertingLac-OR^(1A) as described herein into Gb3-OR^(1A) in a Gb3-synthesisreaction mixture comprising at least the UDP-Gal, an alpha-1,4galactosyltransferase, and the UDP-Gal regeneration enzymes, (iii)mixing the Gb3-synthesis reaction mixture with at least GalNAc, thebeta1,3-N-acetylgalactosaminyltransferase, the N-acetylhexosamine1-kinase, and the N-acetylglucosamine 1-phosphate uridyltransferase toform a Gb4-synthesis reaction mixture, (iv) incubating the Gb4-synthesisreaction mixture under conditions allowing conversion of Gb3-OR^(1A) toGb4-OR^(1A), (v) further incubating the Gb4-synthesis reaction mixturein the presence of a β-1,3-galactosyltransferase under conditionsallowing conversion of the Gb4-OR^(1A) to Gb5-OR^(1A), (vi) mixing theGb5-OR^(1A)-containing reaction mixture with at least fucose, GTP, thealpha1,2-fucosyltransferase, and the L-fucokinase/GDP-fucosepyrophosphorylase to form a Fucosyl-Gb5-OR^(1A) reaction mixture; (vii)incubating the Fucosyl-Gb5-OR^(1A) reaction mixture under conditionsallowing conversion of the Gb5-OR^(1A) to Fucosyl-Gb5-OR^(1A), andoptionally, (viii) isolating the Fucosyl-Gb5-OR^(1A).

In another example, a method for synthesizing Globo H can be performedas follows: (i) producing UDP-Gal from galactose in the presence of theUDP-Gal regeneration enzymes as described herein, (ii) convertingLac-OR^(1A) as described herein into Gb3-OR^(1A) in a Gb3-synthesisreaction mixture comprising at least the UDP-Gal, an alpha-1,4galactosyltransferase, and the UDP-Gal regeneration enzymes, (iii)mixing the Gb3-synthesis reaction mixture with at least GalNAc, thebeta1,3-N-acetylgalactosaminyltransferase, the N-acetylhexosamine1-kinase, and the N-acetylglucosamine 1-phosphate uridyltransferase toform a Gb4-synthesis reaction mixture, (iv) incubating the Gb4-synthesisreaction mixture under conditions allowing conversion of Gb3-OR^(1A) toGb4-OR^(1A); (v) isolating the Gb4-OR^(1A); (vi) mixing the Gb4-OR^(1A)with a beta1,3-galactosyltransferase and the set of UDP-Gal regenerationenzymes to form a Gb5-synthesis reaction mixture; (vii) incubating theGb5-synthesis reaction mixture under conditions allowing conversion ofthe Gb4-OR^(AI) to Gb5-OR^(1A), (viii) mixing the Gb5-synthesis reactionmixture with at least at least fucose, GTP, thealpha1,2-fucosyltransferase, and the L-fucokinase/GDP-fucosepyrophosphorylase to form a Fucosyl-Gb5-OR^(1A) reaction mixture; (ix)incubating the Fucosyl-Gb5-OR^(1A) reaction mixture under conditionsallowing conversion of the Gb5-OR^(1A) to Fucosyl-Gb5-OR^(1A); andoptionally, (x) isolating the Fucosyl-Gb5-OR^(1A).

A method for synthesizing SSEA4 can be performed as follows: (i)producing UDP-Gal from galactose in the presence of the UDP-Galregeneration enzymes as described herein, (ii) converting Lac-OR^(1A) asdescribed herein into Gb3-OR^(1A) in a Gb3-synthesis reaction mixturecomprising at least the UDP-Gal, an alpha-1,4 galactosyltransferase, andthe UDP-Gal regeneration enzymes, (iii) mixing the Gb3-synthesisreaction mixture with at least GalNAc, thebeta1,3-N-acetylgalactosaminyltransferase, the N-acetylhexosamine1-kinase, and the N-acetylglucosamine 1-phosphate uridyltransferase toform a Gb4-synthesis reaction mixture, (iv) incubating the Gb4-synthesisreaction mixture under conditions allowing conversion of Gb3-OR^(1A) toGb4-OR^(1A), (v) further incubating the Gb4-synthesis reaction mixturein the presence of a β-1,3-galactosyltransferase under conditionsallowing conversion of the Gb4-OR^(1A) to Gb5-OR^(1A), (vi) mixing theGb4-synthesis reaction mixture with at least at least Neu5Ac, CTP, thealpha2,3-sialyltransferase, the cytidine monophosphate kinase, and theCMP-sialic acid synthetase to form a Sialyl-Gb5-OR^(1A) reactionmixture; (vii) incubating the Sialyl-Gb5-OR^(1A) reaction mixture underconditions allowing conversion of the Gb5-OR^(1A) to Sialyl-Gb5-OR^(1A);and optionally, (viii) isolating the Sialyl-Gb5-OR^(1A).

Alternatively, a method for synthesizing SSEA4 can be performed asfollows: (i) producing UDP-Gal from galactose in the presence of theUDP-Gal regeneration enzymes as described herein, (ii) convertingLac-OR^(1A) as described herein into Gb3-OR^(1A) in a Gb3-synthesisreaction mixture comprising at least the UDP-Gal, an alpha-1,4galactosyltransferase, and the UDP-Gal regeneration enzymes, (iii)mixing the Gb3-synthesis reaction mixture with at least GalNAc, thebeta1,3-N-acetylgalactosaminyltransferase, the N-acetylhexosamine1-kinase, and the N-acetylglucosamine 1-phosphate uridyltransferase toform a Gb4-synthesis reaction mixture, (iv) incubating the Gb4-synthesisreaction mixture under conditions allowing conversion of Gb3-OR^(1A) toGb4-OR^(1A); (v) isolating the Gb4-OR^(1A); (vi) mixing the Gb4-OR^(1A)with a beta1,3-galactosyltransferase and the set of UDP-Gal regenerationenzymes to form a Gb5-synthesis reaction mixture; (vii) incubating theGb5-synthesis reaction mixture under conditions allowing conversion ofthe Gb4-OR^(1A) to Gb5-OR^(1A); (viii) mixing the Gb5-OR^(1A) with analpha2,3sialyltransferase and a set of CMP-Neu5Ac regeneration enzymesto form a Sialyl-Gb5-synthesis reaction mixture, wherein the set ofCMP-Neu5Ac regeneration enzymes comprises a cytidine monophosphatekinase, a CMP-sialic acid synthetase, a pyruvate kinase, and apyrophosphatase; (ix) incubating the Sialyl-Gb5-synthesis reactionmixture under conditions allowing conversion of the Gb4-OR^(1A) toSialyl-Gb5-OR^(1A); and optionally, (x) isolating theSialyl-Gb5-OR^(1A).

In some embodiments, the method described herein for enzymaticallysynthesizing an oligosaccharide uses Gb3 (e.g., tailed) as the startingmaterial. The method comprises: (i) producing UDP-GalNAc from GalNAc inthe presence of the set of UDP-GalNAc regeneration enzymes as describedabove, and converting Gb3-OR^(1A) into Gb4-OR^(1A) in the presence ofthe UDP-GalNAc and a beta1,3-N-acetylgalactosaminyltransferase, whereinR^(1A) is hydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, or an oxygen protecting group. Examples of R^(1A) include,but are not limited to, hydrogen, allyl, biotin, a ceramide, or anon-hydrogen group (e.g., alkyl) which is further substituted with asubstituted or unsubstituted thio, substituted or unsubstituted amino,carbonyl (e.g., carboxylic acid), azido, alkenyl (e.g., allyl), alkynyl(e.g., propargyl), biotin, or a ceramide group. In certain embodiments,R^(1A) is hydrogen, allyl, substituted alkyl, biotin, or a ceramide.Steps (i) and (ii) can occur in a Gb4-synthesis reaction mixturecomprising GalNAc, PEP, ATP, UTP, the Gb3-OR^(1A), thebeta1,3-N-acetylgalactosaminyltransferase, and the set of UDP-GalNAcregeneration enzymes. The Gb4-OR^(1A) can be isolated if necessary.

The above method can further comprise: (iii) converting the Gb4-OR^(1A)into Gb5-OR^(1A) in the presence of UDP-Gal and abeta1,3-galactosyltransferase, which can be coupled with (iv) producingthe UDP-Gal from galactose in the presence of the set of UDP-Galregeneration enzymes as described herein. (iii) and (iv) can take placein a Gb5-synthesis reaction mixture comprising galactose, PEP, ATP, UTP,the Gb4-OR^(1A), the beta1,3-galactosyltransferase, and the set ofUDP-Gal regeneration enzymes. The resultant Gb5-OR^(1A) can be isolatedfrom the reaction mixture.

In one example, the Gb5-OR^(1A) is then converted intoFucosyl-Gb5-OR^(1A) as follows: (v) converting the Gb5-OR^(1A) intoFucosyl-Gb5-OR^(1A) in the presence of GDP-Fuc and analpha1,2-fucosyltransferase, which can be coupled with (vi) producingthe GDP-Fuc from fucose in the presence of the set of GDP-Fucregeneration enzymes described herein. Steps (v) and (vi) can be carriedout in a Fucosyl-Gb5-synthesis reaction mixture comprising fucose, ATP,GTP, PEP, the Gb5-OR^(1A), the alpha1,2-fucosyltransferase, and the setof GDP-Fuc regeneration enzymes. When desired, the Fucosyl-Gb5-synthesisreaction mixture is prepared by mixing the Gb5-synthesis reactionmixture with at least fucose, GTP, the alpha1,2-fucosyltransferase, andthe L-fucokinase/GDP-fucose pyrophosphorylase. The method can furthercomprise isolating the Fucosyl-Gb5-OR^(1A).

In another example, the Gb5-OR^(1A) is then converted intoSialyl-Gb5-OR^(1A) as follows: (vii) converting the Gb5-OR^(1A) intoSialyl-Gb5-OR^(1A) in the presence of CMP-Neu5Ac and an alpha2,3-sialyltransferase, which can be coupled with (viii) producing theCMP-Neu5Ac from Neu5Ac in the presence of the set of CMP-Neu5Acregeneration enzymes described herein. Steps (vii) and (viii) can occurin a Sialyl-Gb5-synthesis reaction mixture comprising Neu5Ac, CTP, PEP,the Gb5-OR^(1A), the alpha 2,3-sialyltransferase, and the set ofCMP-Neu5Ac regeneration enzymes. In some instances, theSialyl-Gb5-synthesis reaction mixture is prepared by mixing theGb5-synthesis reaction mixture with at least Neu5Ac, CTP, the alpha2,3-sialyltransferase, the cytidine monophosphate kinase, and theCMP-sialic acid synthetase. The resultant Sialyl-Gb5-OR^(1A) can beisolated from the reaction mixture.

In yet other embodiments, the methods described herein relate tosynthesizing oligosaccharides, using Gb4 (e.g., tailed) as a startingmaterial. Such a method comprises: (i) producing UDP-Gal from galactosein the presence of the set of UDP-Gal regeneration enzymes describedherein, and (ii) converting Gb4-OR^(1A) into Gb5-OR^(1A) in the presenceof UDP-Gal and a beta1,3-galactosyltransferase, wherein R^(1A) ishydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, or an oxygen protecting group. Examples of R^(1A) include,but are not limited, to hydrogen, allyl, biotin, a ceramide, or anon-hydrogen group (e.g., alkyl) which is further substituted with asubstituted or unsubstituted thio, substituted or unsubstituted amino,carbonyl (e.g., carboxylic acid), azido, alkenyl (e.g., allyl), alkynyl(e.g., propargyl), biotin, or a ceramide group. In certain embodiments,R^(1A) is hydrogen, allyl, substituted alkyl, biotin, or a ceramide. Inthis method, steps (i) and (ii) can occur in a Gb5-synthesis reactionmixture comprising galactose, PEP, ATP, UTP, the Gb4-OR^(1A), thebeta1,3-galactosyltransferase, and the set of UDP-Gal regenerationenzymes. Alternatively or in addition, the Gb5-OR^(1A) thus produced canbe isolated.

The above method can further comprise: (iii) converting the Gb5-OR^(1A)into Fucosyl-Gb5-OR^(1A) in the presence of GDP-Fuc and analpha1,2-fucosyltransferase, which can be coupled with (iv) producingthe GDP-Fuc from fucose in the presence of the set of GDP-Fucregeneration enzymes, which is also described herein. Steps (iii) and(iv) can take place in a Fucosyl-Gb5-synthesis reaction mixturecomprising fucose, ATP, GTP, PEP, the Gb5-OR^(1A), thealpha1,2-fucosyltransferase, and the set of GDP-Fuc regenerationenzymes. The Fucosyl-Gb5-synthesis reaction mixture is prepared bymixing the Gb5-synthesis reaction mixture with at least fucose, GTP, thealpha1,2-fucosyltransferase, and the L-fucokinase/GDP-fucosepyrophosphorylase. The resultant Fucosyl-Gb5-OR^(1A) can be isolatedfrom the reaction mixture.

Alternatively, the above method can further comprise: (v) converting theGb5-OR^(1A) into Sialyl-Gb5-OR^(1A) in the presence of CMP-Neu5Ac and analpha 2,3-sialyltransferase, which can be coupled with (v) producing theCMP-Neu5Ac from Neu5Ac in the presence of the set of CMP-Neu5Acregeneration enzymes described herein. Steps (v) and (vi) can occur in aSialyl-Gb5-synthesis reaction mixture comprising Neu5Ac, CTP, PEP, theGb5-OR^(1A), the alpha 2,3-sialyltransferase, and the set of CMP-Neu5Acregeneration enzymes. The Sialyl-Gb5-synthesis reaction mixture isprepared by mixing the Gb5-synthesis reaction mixture with at leastNeu5Ac, CTP, the alpha 2,3-sialyltransferase, the cytidine monophosphatekinase, and the CMP-sialic acid synthetase. The Sialyl-Gb5-OR^(1A)produced in this method can be isolated from the reaction mixture.

In some other embodiments, the methods described herein relate tosynthesis of a Fucosyl-Gb5 oligosaccharide (Globo H) from Gb5. Themethod comprising: (i) producing GDP-Fuc from fucose in the presence ofthe set of GDP-Fuc regeneration enzymes described herein, (ii)converting Gb5-OR^(1A) into Fucosyl-Gb5-OR^(1A) in the presence of theGDP-Fuc and an alpha1,2-fucosyltransferase, and, optionally, (iii)isolating the Fucosyl-Gb5-OR^(1A) Steps (i) and (ii) can occur in aFucosyl-Gb5-synthesis reaction mixture comprising fucose, ATP, GTP, PEP,the Gb5-OR^(1A), the alpha1,2-fucosyltransferase, and the set of GDP-Fucregeneration enzymes.

In some other embodiments, the methods described herein relate tosynthesis of a Sialyl-Gb5 oligosaccharide (Globo H) from Gb5. The methodcomprises: (i) producing CMP-Neu5Ac from Neu5Ac in the presence of theset of CMP-Neu5Ac regeneration enzymes described herein, (ii) convertingGb5-OR^(1A) into Sialyl-Gb5-OR^(1A) in the presence of CMP-Neu5Ac and analpha 2,3-sialyltransferase, and, optionally, (iii) isolating theSialyl-Gb5-OR^(1A). Steps (i) and (ii) can take place in aSialyl-Gb5-synthesis reaction mixture comprising Neu5Ac, CTP, PEP, theGb5-OR, the alpha 2,3-sialyltransferase, and the set of CMP-Neu5Acregeneration enzymes.

In any of the synthesis methods described herein, either at least one ofthe involved enzymes or at least one of the substrates of each reaction(e.g., lactose, Gb3, Gb4, or Gb5) can be immobilized on a supportmember.

Another aspect of the present disclosure features enzymatic reactors forsynthesizing oligosaccharides using the methods described herein. Suchan enzymatic reactor can comprise one or more of the following reactionchambers:

(a) a reaction chamber for synthesizing Gb3-OR^(1A), wherein the chambercomprises an alpha1,4-galactosyltransferase, and a set of UDP-Galregeneration enzymes, which comprises a galactokinase, a UDP-sugarpyrophosphorylase, a pyruvate kinase, and optionally a pyrophosphatase;

(b) a reaction chamber for synthesizing Gb4-OR^(1A), wherein the chambercomprises a beta1,3-N-acetylgalactosaminyltransferase and a set ofUDP-GalNAc regeneration enzymes, which comprises an N-acetylhexosamine1-kinase, an N-acetylglucosamine 1-phosphate uridylyltransferase, apyruvate kinase, and optionally a pyrophosphatase;

(c) a reaction chamber for synthesizing Gb5-OR^(1A), wherein the chambercomprises a beta1,3-galactosyltransferase, and the set of UDP-Galregeneration enzymes;

(d) a reaction chamber for synthesizing Fucosyl-Gb5-OR^(1A), wherein thechamber comprises an alpha1,2-fucosyltransferase and a set of GDP-Fucregeneration enzymes, which comprises an L-fucokinase/GDP-fucosepyrophosphorylase, a pyruvate kinase, and optionally a pyrophosphatase;and

(e) a reaction chamber for synthesizing Sialyl-Gb5-OR^(1A), wherein thechamber comprises an alpha2,3-sialyltransferase and a set of CMP-Neu5Acregeneration enzymes, which comprises a cytidine monophosphate kinase, aCMP-sialic acid synthetase, a pyruvate kinase, and optionally apyrophosphatase.

In some examples, the enzymatic reactor comprises reaction chambers: (a)and (b); (a), (b), and (c); (a), (b), (c), and (d); (a), (b), (c), and(e); (b) and (c); (b), (c), and (d); (b), (c), and (e); (c) and (d); or(c) and (e).

In another example, the enzymatic reactor described herein comprises areaction chamber that comprises a galactosyltransferase (e.g., analpha1,4-galactosyltransferase, a beta1,4-galactosyltransferase, analpha1,3-galactosyltransferase, or a beta1,3-galactosyltransferase) anda set of UDP-Gal regeneration enzymes as described herein, which maycomprise a galactokinase, an UDP pyrophosphorylase, a pyruvate kinase,and optionally a pyrophosphatase.

In any of the reaction chambers, one or more of the enzymes can beimmobilized on a support member. In some examples, one or more of theset of UDP-Gal regeneration enzymes, the set of UDP-GalNAc regenerationenzymes, the set of GDP-Fuc regeneration enzymes, and the set ofCMP-Neu5Ac regeneration enzymes are each immobilized on a supportmember. In other examples, all of the enzymes in a reaction chamber areimmobilized on a support member.

Also within the scope of the present disclosure are oligosaccharidesobtained from any of the synthesis methods described herein.

The details of one or more embodiments of the invention are set forth inthe Detailed Description of Certain Embodiments, as described below.Other features, objects, and advantages of the invention will beapparent from the Definitions, Drawings, Examples, and Claims.

CHEMICAL DEFINITIONS

Definitions of specific functional groups and chemical terms aredescribed in more detail below. The chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed., inside cover, andspecific functional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in OrganicChemistry, Thomas Sorrell, University Science Books, Sausalito, 1999;Smith and March March's Advanced Organic Chemistry, 5^(th) Edition, JohnWiley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; and Carruthers,Some Modern Methods of Organic Synthesis, 3^(rd) Edition, CambridgeUniversity Press, Cambridge, 1987.

Compounds described herein can comprise one or more asymmetric centers,and thus can exist in various stereoisomeric forms, e.g., enantiomersand/or diastereomers. For example, the compounds described herein can bein the form of an individual enantiomer, diastereomer or geometricisomer, or can be in the form of a mixture of stereoisomers, includingracemic mixtures and mixtures enriched in one or more stereoisomer.Isomers can be isolated from mixtures by methods known to those skilledin the art, including chiral high pressure liquid chromatography (HPLC)and the formation and crystallization of chiral salts; or preferredisomers can be prepared by asymmetric syntheses. See, for example,Jacques et al., Enantiomers, Racemates and Resolutions (WileyInterscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977);Eliel, E. L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY,1962); and Wilen, S. H. Tables of Resolving Agents and OpticalResolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, NotreDame, Ind. 1972). The invention additionally encompasses compounds asindividual isomers substantially free of other isomers, andalternatively, as mixtures of various isomers.

When a range of values is listed, it is intended to encompass each valueand sub-range within the range. For example “C₁₋₆ alkyl” is intended toencompass, C₁, C₂, C₃, C₄, C₅, C₆, C₁₋₆, C₁₋₅, C₁₋₄, C₁₋₃, C₁₋₂, C₂₋₆,C₂₋₅, C₂₋₄, C₂₋₃, C₃₋₆, C₃₋₅, C₃₋₄, C₄₋₆, C₄₋₅, and C₅₋₆ alkyl.

As used herein, “alkyl” refers to a radical of a straight-chain orbranched saturated hydrocarbon group having from 1 to 30 carbon atoms(“C₁₋₃₀ alkyl”). In some embodiments, an alkyl group has 1 to 20 carbonatoms (“C₁₋₂₀ alkyl”). In some embodiments, an alkyl group has 1 to 10carbon atoms (“C₁₋₁₀ alkyl”). In some embodiments, an alkyl group has 1to 9 carbon atoms (“C₁₋₉ alkyl”). In some embodiments, an alkyl grouphas 1 to 8 carbon atoms (“C₁₋₈ alkyl”). In some embodiments, an alkylgroup has 1 to 7 carbon atoms (“C₁₋₇ alkyl”). In some embodiments, analkyl group has 1 to 6 carbon atoms (“C₁₋₆ alkyl”). In some embodiments,an alkyl group has 1 to 5 carbon atoms (“C₁₋₅ alkyl”). In someembodiments, an alkyl group has 1 to 4 carbon atoms (“C₁₋₄ alkyl”). Insome embodiments, an alkyl group has 1 to 3 carbon atoms (“C₁₋₃ alkyl”).In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C₁₋₂alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“C₁alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms(“C₂₋₆alkyl”). Examples of C₁₋₆ alkyl groups include methyl (C₁), ethyl(C₂), n-propyl (C₃), isopropyl (C3), n-butyl (C₄), tert-butyl (C₄),sec-butyl (C₄), iso-butyl (C₄), n-pentyl (C₅), 3-pentanyl (C₅), amyl(C₅), neopentyl (C₅), 3-methyl-2-butanyl (C₅), tertiary amyl (C₅), andn-hexyl (C₆). Additional examples of alkyl groups include n-heptyl (C₇),n-octyl (C₈) and the like. Unless otherwise specified, each instance ofan alkyl group is independently unsubstituted (an “unsubstituted alkyl”)or substituted (a “substituted alkyl”) with one or more substituents. Incertain embodiments, the alkyl group is an unsubstituted C₁₋₁₀ alkyl(e.g., —CH₃). In certain embodiments, the alkyl group is a substitutedC₁₋₁₀ alkyl.

As used herein, “alkenyl” or “alkene” refers to a radical of astraight-chain or branched hydrocarbon group having from 2 to 30 carbonatoms and one or more double bonds (e.g., 1, 2, 3, or 4 double bonds).In some embodiments, an alkenyl group has 2 to 20 carbon atoms (“C₂₋₂₀alkenyl”). In some embodiments, an alkenyl group has 2 to 10 carbonatoms (“C₂₋₁₀ alkenyl”). In some embodiments, an alkenyl group has 2 to9 carbon atoms (“C₂₋₉ alkenyl”). In some embodiments, an alkenyl grouphas 2 to 8 carbon atoms (“C₂₋₈ alkenyl”). In some embodiments, analkenyl group has 2 to 7 carbon atoms (“C₂₋₇alkenyl”). In someembodiments, an alkenyl group has 2 to 6 carbon atoms (“C₂₋₆ alkenyl”).In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C₂₋₅alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms(“C₂₋₄ alkenyl”). In some embodiments, an alkenyl group has 2 to 3carbon atoms (“C₂₋₃ alkenyl”). In some embodiments, an alkenyl group has2 carbon atoms (“C₂ alkenyl”). The one or more carbon-carbon doublebonds can be internal (such as in 2-butenyl) or terminal (such as in1-butenyl). Examples of C₂₋₄ alkenyl groups include ethenyl (C₂),1-propenyl (“allyl”, C₃), 2-propenyl (C₃), 1-butenyl (C₄), 2-butenyl(C₄), butadienyl (C₄), and the like. Examples of C₂₋₆ alkenyl groupsinclude the aforementioned C₂₋₄ alkenyl groups as well as pentenyl (C₅),pentadienyl (C₅), hexenyl (C₆), and the like. Additional examples ofalkenyl include heptenyl (C₇), octenyl (C₈), octatrienyl (C₆), and thelike. Unless otherwise specified, each instance of an alkenyl group isindependently unsubstituted (an “unsubstituted alkenyl”) or substituted(a “substituted alkenyl”) with one or more substituents. In certainembodiments, the alkenyl group is an unsubstituted C₂₋₁₀ alkenyl. Incertain embodiments, the alkenyl group is a substituted C₂₋₁₀ alkenyl.

As used herein, “alkynyl” or “alkyne” refers to a radical of astraight-chain or branched hydrocarbon group having from 2 to 30 carbonatoms and one or more triple bonds (e.g., 1, 2, 3, or 4 triple bonds)(“C₂₋₁₀ alkynyl”). In some embodiments, an alkynyl group has 2 to 20carbon atoms (“C₂₋₂₀ alkynyl”). In some embodiments, an alkynyl grouphas 2 to 10 carbon atoms (“C₂₋₁₀ alkynyl”). In some embodiments, analkynyl group has 2 to 9 carbon atoms (“C₂₋₉ alkynyl”). In someembodiments, an alkynyl group has 2 to 8 carbon atoms (“C₂₋₈ alkynyl”).In some embodiments, an alkynyl group has 2 to 7 carbon atoms (“C₂₋₇alkynyl”). In some embodiments, an alkynyl group has 2 to 6 carbon atoms(“C₂₋₆ alkynyl”). In some embodiments, an alkynyl group has 2 to 5carbon atoms (“C₂₋₅ alkynyl”). In some embodiments, an alkynyl group has2 to 4 carbon atoms (“C₂₋₄ alkynyl”). In some embodiments, an alkynylgroup has 2 to 3 carbon atoms (“C₂₋₃ alkynyl”). In some embodiments, analkynyl group has 2 carbon atoms (“C₂ alkynyl”). The one or morecarbon-carbon triple bonds can be internal (such as in 2-butynyl) orterminal (such as in 1-butynyl). Examples of C₂₋₄ alkynyl groupsinclude, without limitation, ethynyl (C₂), 1-propynyl (C₃), 2-propynyl(C₃), 1-butynyl (C₄), 2-butynyl (C₄), and the like. Examples of C2,alkenyl groups include the aforementioned C₂₋₄ alkynyl groups as well aspentynyl (C₅), hexynyl (C₆), and the like. Additional examples ofalkynyl include heptynyl (C₇), octynyl (C₈), and the like. Unlessotherwise specified, each instance of an alkynyl group is independentlyunsubstituted (an “unsubstituted alkynyl”) or substituted (a“substituted alkynyl”) with one or more substituents. In certainembodiments, the alkynyl group is an unsubstituted C₂₋₁₀ alkynyl. Incertain embodiments, the alkynyl group is a substituted C₂₋₁₀ alkynyl.

As used herein, “carbocyclyl” refers to a radical of a non-aromaticcyclic hydrocarbon group having from 3 to 10 ring carbon atoms (“C₃₋₁₀carbocyclyl”) and zero heteroatoms in the non-aromatic ring system. Insome embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms(“C₃₋₈ carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to7 ring carbon atoms (“C₃₋₇ carbocyclyl”). In some embodiments, acarbocyclyl group has 3 to 6 ring carbon atoms (“C₃₋₆ carbocyclyl”). Insome embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms(“C₅₋₁₀ carbocyclyl”). Exemplary C₃₋₆ carbocyclyl groups include,without limitation, cyclopropyl (C₃), cyclopropenyl (C₃), cyclobutyl(C₄), cyclobutenyl (C₄), cyclopentyl (C₅), cyclopentenyl (C₅),cyclohexyl (C₆), cyclohexenyl (C₆), cyclohexadienyl (C₆), and the like.Exemplary C₃₋₈ carbocyclyl groups include, without limitation, theaforementioned C₃₋₆ carbocyclyl groups as well as cycloheptyl (C₇),cycloheptenyl (C₇), cycloheptadienyl (C₇), cycloheptatrienyl (C₇),cyclooctyl (C₅), cyclooctenyl (C₅), bicyclo[2.2.1]heptanyl (C₇),bicyclo[2.2.2]octanyl (C₈), and the like. Exemplary C₃₋₁₀ carbocyclylgroups include, without limitation, the aforementioned C₃₋₈ carbocyclylgroups as well as cyclononyl (C₉), cyclononenyl (C₉), cyclodecyl (C₁₀),cyclodecenyl (C₁₀), octahydro-1H-indenyl (C₉), decahydronaphthalenyl(C₁₀), spiro[4.5]decanyl (C₁₀), and the like. As the foregoing examplesillustrate, in certain embodiments, the carbocyclyl group is eithermonocyclic (“monocyclic carbocyclyl”) or polycyclic (e.g., containing afused, bridged or spiro ring system such as a bicyclic system (“bicycliccarbocyclyl”) or tricyclic system (“tricyclic carbocyclyl”)) and can besaturated or can contain one or more carbon-carbon double or triplebonds. “Carbocyclyl” also includes ring systems wherein the carbocyclylring, as defined above, is fused with one or more aryl or heteroarylgroups wherein the point of attachment is on the carbocyclyl ring, andin such instances, the number of carbons continue to designate thenumber of carbons in the carbocyclic ring system. Unless otherwisespecified, each instance of a carbocyclyl group is independentlyunsubstituted (an “unsubstituted carbocyclyl”) or substituted (a“substituted carbocyclyl”) with one or more substituents. In certainembodiments, the carbocyclyl group is an unsubstituted C₃₋₁₀carbocyclyl. In certain embodiments, the carbocyclyl group is asubstituted C₃₋₁₀ carbocyclyl.

In some embodiments, “carbocyclyl” is a monocyclic, saturatedcarbocyclyl group having from 3 to 10 ring carbon atoms (“C₃₋₁₀cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ringcarbon atoms (“C₃₋₈ cycloalkyl”). In some embodiments, a cycloalkylgroup has 3 to 6 ring carbon atoms (“C₃₋₆ cycloalkyl”). In someembodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C₅₋₆cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ringcarbon atoms (“C₅₋₁₀ cycloalkyl”). Examples of C₅₋₆ cycloalkyl groupsinclude cyclopentyl (C₅) and cyclohexyl (C₅). Examples of C₃₋₆cycloalkyl groups include the aforementioned C₅₋₆ cycloalkyl groups aswell as cyclopropyl (C₃) and cyclobutyl (C₄). Examples of C₃₋₈cycloalkyl groups include the aforementioned C₃₋₆ cycloalkyl groups aswell as cycloheptyl (C₇) and cyclooctyl (C₈). Unless otherwisespecified, each instance of a cycloalkyl group is independentlyunsubstituted (an “unsubstituted cycloalkyl”) or substituted (a“substituted cycloalkyl”) with one or more substituents. In certainembodiments, the cycloalkyl group is an unsubstituted C₃₋₁₀ cycloalkyl.In certain embodiments, the cycloalkyl group is a substituted C₃₋₁₀cycloalkyl.

As used herein, “heterocyclyl” refers to a radical of a 3- to14-membered non-aromatic ring system having ring carbon atoms and 1 to 4ring heteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“3-14 membered heterocyclyl”). Inheterocyclyl groups that contain one or more nitrogen atoms, the pointof attachment can be a carbon or nitrogen atom, as valency permits. Aheterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”)or polycyclic (e.g., a fused, bridged or spiro ring system such as abicyclic system (“bicyclic heterocyclyl”) or tricyclic system(“tricyclic heterocyclyl”)), and can be saturated or can contain one ormore carbon-carbon double or triple bonds. Heterocyclyl polycyclic ringsystems can include one or more heteroatoms in one or both rings.“Heterocyclyl” also includes ring systems wherein the heterocyclyl ring,as defined above, is fused with one or more carbocyclyl groups whereinthe point of attachment is either on the carbocyclyl or heterocyclylring, or ring systems wherein the heterocyclyl ring, as defined above,is fused with one or more aryl or heteroaryl groups, wherein the pointof attachment is on the heterocyclyl ring, and in such instances, thenumber of ring members continue to designate the number of ring membersin the heterocyclyl ring system. Unless otherwise specified, eachinstance of heterocyclyl is independently unsubstituted (an“unsubstituted heterocyclyl”) or substituted (a “substitutedheterocyclyl”) with one or more substituents. In certain embodiments,the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl.In certain embodiments, the heterocyclyl group is a substituted 3-14membered heterocyclyl.

In some embodiments, a heterocyclyl group is a 5-10 memberednon-aromatic ring system having ring carbon atoms and 1-4 ringheteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“5-10 membered heterocyclyl”). In someembodiments, a heterocyclyl group is a 5-8 membered non-aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms, wherein eachheteroatom is independently selected from nitrogen, oxygen, and sulfur(“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl groupis a 5-6 membered non-aromatic ring system having ring carbon atoms and1-4 ring heteroatoms, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In someembodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclylhas 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

Exemplary 3-membered heterocyclyl groups containing 1 heteroatominclude, without limitation, azirdinyl, oxiranyl, thiorenyl. Exemplary4-membered heterocyclyl groups containing 1 heteroatom include, withoutlimitation, azetidinyl, oxetanyl and thietanyl. Exemplary 5-memberedheterocyclyl groups containing 1 heteroatom include, without limitation,tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl,dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl and pyrrolyl-2,5-dione.Exemplary 5-membered heterocyclyl groups containing 2 heteroatomsinclude, without limitation, dioxolanyl, oxathiolanyl and dithiolanyl.Exemplary 5-membered heterocyclyl groups containing 3 heteroatomsinclude, without limitation, triazolinyl, oxadiazolinyl, andthiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing 1heteroatom include, without limitation, piperidinyl, tetrahydropyranyl,dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groupscontaining 2 heteroatoms include, without limitation, piperazinyl,morpholinyl, dithianyl, dioxanyl. Exemplary 6-membered heterocyclylgroups containing 2 heteroatoms include, without limitation,triazinanyl. Exemplary 7-membered heterocyclyl groups containing 1heteroatom include, without limitation, azepanyl, oxepanyl andthiepanyl. Exemplary 8-membered heterocyclyl groups containing 1heteroatom include, without limitation, azocanyl, oxecanyl andthiocanyl. Exemplary bicyclic heterocyclyl groups include, withoutlimitation, indolinyl, isoindolinyl, dihydrobenzofuranyl,dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl,tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl,octahydroisochromenyl, decahydronaphthyridinyl,decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl,phthalimidyl, naphthalimidyl, chromanyl, chromenyl,1H-benzo[e][1,4]diazepinyl, 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl,5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl,5,7-dihydro-4H-thieno[2,3-c]pyranyl,2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl,4,5,6,7-tetrahydro-1H-pyrrolo[2,3-b]pyridinyl,4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl,4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl,1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like.

As used herein, “aryl” refers to a radical of a monocyclic or polycyclic(e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6,10, or 14 π electrons shared in a cyclic array) having 6-14 ring carbonatoms and zero heteroatoms provided in the aromatic ring system (“C₆₋₁₄aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“C₆aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ringcarbon atoms (“C₁₀ aryl”; e.g., naphthyl such as 1-naphthyl and2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms(“C₁₄ aryl”; e.g., anthracyl). “Aryl” also includes ring systems whereinthe aryl ring, as defined above, is fused with one or more carbocyclylor heterocyclyl groups wherein the radical or point of attachment is onthe aryl ring, and in such instances, the number of carbon atomscontinue to designate the number of carbon atoms in the aryl ringsystem. Unless otherwise specified, each instance of an aryl group isindependently unsubstituted (an “unsubstituted aryl”) or substituted (a“substituted aryl”) with one or more substituents. In certainembodiments, the aryl group is an unsubstituted C₆₋₁₄ aryl. In certainembodiments, the aryl group is a substituted C₆₋₁₄ aryl.

As used herein, “heteroaryl” refers to a radical of a 5-14 memberedmonocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ringsystem (e.g., having 6, 10, or 14 π electrons shared in a cyclic array)having ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen and sulfur (“5-14 membered heteroaryl”). Inheteroaryl groups that contain one or more nitrogen atoms, the point ofattachment can be a carbon or nitrogen atom, as valency permits.Heteroaryl polycyclic ring systems can include one or more heteroatomsin one or both rings. “Heteroaryl” includes ring systems wherein theheteroaryl ring, as defined above, is fused with one or more carbocyclylor heterocyclyl groups wherein the point of attachment is on theheteroaryl ring, and in such instances, the number of ring memberscontinue to designate the number of ring members in the heteroaryl ringsystem. “Heteroaryl” also includes ring systems wherein the heteroarylring, as defined above, is fused with one or more aryl groups whereinthe point of attachment is either on the aryl or heteroaryl ring, and insuch instances, the number of ring members designates the number of ringmembers in the fused polycyclic (aryl/heteroaryl) ring system.Polycyclic heteroaryl groups wherein one ring does not contain aheteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) thepoint of attachment can be on either ring, i.e., either the ring bearinga heteroatom (e.g., 2-indolyl) or the ring that does not contain aheteroatom (e.g., 5-indolyl).

In some embodiments, a heteroaryl group is a 5-10 membered aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-8 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-6 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In someembodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unlessotherwise specified, each instance of a heteroaryl group isindependently unsubstituted (an “unsubstituted heteroaryl”) orsubstituted (a “substituted heteroaryl”) with one or more substituents.In certain embodiments, the heteroaryl group is an unsubstituted 5-14membered heteroaryl. In certain embodiments, the heteroaryl group is asubstituted 5-14 membered heteroaryl.

Exemplary 5-membered heteroaryl groups containing 1 heteroatom include,without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary5-membered heteroaryl groups containing 2 heteroatoms include, withoutlimitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, andisothiazolyl. Exemplary 5-membered heteroaryl groups containing 3heteroatoms include, without limitation, triazolyl, oxadiazolyl, andthiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4heteroatoms include, without limitation, tetrazolyl. Exemplary6-membered heteroaryl groups containing 1 heteroatom include, withoutlimitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing2 heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, andpyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4heteroatoms include, without limitation, triazinyl and tetrazinyl,respectively. Exemplary 7-membered heteroaryl groups containing 1heteroatom include, without limitation, azepinyl, oxepinyl, andthiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, withoutlimitation, indolyl, isoindolyl, indazolyl, benzotriazolyl,benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl,benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl,benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, andpurinyl. Exemplary 6,6-bicyclic heteroaryl groups include, withoutlimitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl,cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplarytricyclic heteroaryl groups include, without limitation,phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl,phenoxazinyl and phenazinyl.

As used herein, the term “partially unsaturated” refers to a ring moietythat includes at least one double or triple bond. The term “partiallyunsaturated” is intended to encompass rings having multiple sites ofunsaturation, but is not intended to include aromatic groups (e.g., arylor heteroaryl moieties) as herein defined.

As used herein, the term “saturated” refers to a ring moiety that doesnot contain a double or triple bond, i.e., the ring contains all singlebonds.

Alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl groups, as definedherein, are optionally substituted (e.g., “substituted” or“unsubstituted” alkyl, “substituted” or “unsubstituted” alkenyl,“substituted” or “unsubstituted” alkynyl, “substituted” or“unsubstituted” heteroalkyl, “substituted” or “unsubstituted”heteroalkenyl, “substituted” or “unsubstituted” heteroalkynyl,“substituted” or “unsubstituted” carbocyclyl, “substituted” or“unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or“substituted” or “unsubstituted” heteroaryl group). In general, the term“substituted”, whether preceded by the term “optionally” or not, meansthat at least one hydrogen present on a group (e.g., a carbon ornitrogen atom) is replaced with a permissible substituent, e.g., asubstituent which upon substitution results in a stable compound, e.g.,a compound which does not spontaneously undergo transformation such asby rearrangement, cyclization, elimination, or other reaction. Unlessotherwise indicated, a “substituted” group has a substituent at one ormore substitutable positions of the group, and when more than oneposition in any given structure is substituted, the substituent iseither the same or different at each position. The term “substituted” iscontemplated to include substitution with all permissible substituentsof organic compounds, any of the substituents described herein thatresults in the formation of a stable compound. The present inventioncontemplates any and all such combinations in order to arrive at astable compound. For purposes of this invention, heteroatoms such asnitrogen may have hydrogen substituents and/or any suitable substituentas described herein which satisfy the valencies of the heteroatoms andresults in the formation of a stable moiety.

Exemplary carbon atom substituents include, but are not limited to,halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(aa), —ON(R^(bb))₂,—N(R^(bb))₂, —N(R^(bb))₃ ⁺X⁻, —N(OR^(cc))R^(bb), —SH, —SR^(aa),—SSR^(cc), —C(═O)R^(aa), —CO₂H, —CHO, —C(OR^(cc))₂, —CO₂R^(aa),—OC(═O)R^(aa), —OCO₂R^(aa), —C(═O)N(R^(bb))₂, —OC(═O)N(R^(bb))₂,—NR^(bb)C(═O)R^(aa), —NR^(bb)CO₂R^(aa), —NR^(bb)C(═O)N(R^(bb))₂,—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa), —OC(═NR^(bb))R^(aa),—OC(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂, —OC(═NR^(bb))N(R^(bb))₂,—NR^(bb)C(═NR^(bb))N(R^(bb)), —C(═O)NR^(bb)SO₂R^(aa), —NR^(bb)SO₂R^(aa),—SO₂N(R^(bb))₂, —SO₂R^(aa), —SO₂OR^(aa), —OSO₂R^(aa), —S(═O)R^(aa),—OS(═O)R^(aa), —Si(R^(aa))₃, —OSi(R^(aa))₃—C(═S)N(R^(bb))₂,—C(═O)SR^(aa), —C(═S)SR^(aa), —SC(═S)SR^(aa), —SC(═O)SR^(aa),—OC(═O)SR^(aa), —SC(═O)OR^(aa), —SC(═O)R^(aa), —P(═O)₂R^(aa),—OP(═O)₂R^(aa), —P(═O)(R^(aa))₂, —OP(═O)(R^(aa))₂, —OP(═O)(OR^(aa))₂,—P(═O)₂N(R^(bb))₂, —OP(═O)₂N(R^(aa))₂, —P(═O)(NR^(bb))₂,—OP(═O)(NR^(bb))₂, —NR^(bb)P(═O)(OR^(cc))₂, —NR^(bb)P(═O)(NR^(bb))₂,—P(R^(cc))₂, —P(R^(cc))₃, —OP(R^(cc))₂, —OP(R^(aa))₃, —B(R^(aa))₂,—B(OR^(cc))₂, —BR(OR^(cc)), C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, C₁₋₁₀ heteroalkyl, C₂₋₁₀ heteroalkenyl, C₂₋₁₀heteroalkynyl, C₃₋₁₄ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl, wherein each alkyl, alkenyl,alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl,heterocyclyl, aryl, and heteroaryl is independently substituted with 0,1, 2, 3, 4, or 5 R^(dd) groups;

or two geminal hydrogens on a carbon atom are replaced with the group═O, ═S, ═NN(R^(bb))₂, ═NNR^(bb)C(═O)R^(aa), ═NNR^(bb)C(═O)OR^(aa),═NNR^(bb)S(═O)₂R^(aa), ═NR^(bb), or ═NOR^(cc);

each instance of R^(aa) is, independently, selected from C₁₋₁₀ alkyl,C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₁₋₁₀ heteroalkyl,C₂₋₁₀ heteroalkenyl, C₂₋₁₀ heteroalkynyl, C₃₋₁₀ carbocyclyl, 3-14membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or twoR^(aa) groups are joined to form a 3-14 membered heterocyclyl or 5-14membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl,aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or5 R^(dd) groups;

each instance of R^(bb) is, independently, selected from hydrogen, —OH,—OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa), —C(═O)N(R^(cc))₂, —CO₂R^(aa),—SO₂R, —C(═NR)OR^(aa), —C(═NR^(aa))N(R^(cc))₂, —SO₂N(R^(cc))₂,—SO₂R^(aa), —SO₂OR^(aa), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc),—C(═S)SR^(cc), —P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)N(R^(cc))₂,—P(═O)(NR^(aa))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, C₁₋₁₀ heteroalkyl, C₂₋₁₀ heteroalkenyl, C₂₋₁₀ heteroalkynyl,C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14membered heteroaryl, or two R^(bb) groups are joined to form a 3-14membered heterocyclyl or 5-14 membered heteroaryl ring, wherein eachalkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

each instance of R^(cc) is, independently, selected from hydrogen, C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₁₋₁₀heteroalkyl, C₂₋₁₀ heteroalkenyl, C₂₋₁₀ heteroalkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl, or two R^(cc) groups are joined to form a 3-14 memberedheterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl,alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

each instance of R^(dd) is, independently, selected from halogen, —CN,—NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(ee), —ON(R^(ff))₂, —N(R^(ff))₂,—N(R^(ff))₃ ⁺X⁻, —N(OR^(ee))R^(ff), —SH, —SR^(ee), —SSR^(ee),—C(═O)R^(ee), —CO₂H, —CO₂R^(ee), —OC(═O)R^(ee), —OCO₂R^(ee),—C(═O)N(R^(ff))₂, —OC(═O)N(R^(ff))₂, —NR^(ff)C(═O)R^(ee),—NR^(ff)CO₂R^(ee), —NR^(ff)C(═O)N(R^(ff))₂, —C(═NR)OR^(ee),—OC(═NR^(ff))R^(ee), —OC(═NR^(ff))OR^(ee), —C(═NR^(ff))N(R^(ff))₂,—OC(═NR^(ff))N(R^(ff))₂, —NR^(ff)C(═NR^(ff))N(R^(ff))₂,—NR^(ff)SO₂R^(ee), —SO₂N(R^(ff))₂, —SO₂R^(ee), —SO₂OR^(ee), —OSO₂R^(ee),—S(═O)R^(ee), —Si(R^(ee))₃, —OSi(R^(ee))₃, —C(═S)N(R^(ff))₂,—C(═O)SR^(ee), —C(═S)SR^(ee), —SC(═S)SR^(ee), —P(═O)₂R^(ee),—P(═O)(R^(ee))₂, —OP(═O)(R^(ee))₂, —OP(═O)(OR^(ee))₂, C₁₋₆ alkyl, C₁₋₆perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ heteroalkyl, C₂₋₆heteroalkenyl, C₂₋₆heteroalkynyl, C₃₋₁₀ carbocyclyl, 3-10 memberedheterocyclyl, C₆₋₁₀ aryl, 5-10 membered heteroaryl, wherein each alkyl,alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups, or two geminalR^(dd) substituents can be joined to form ═O or ═S;

each instance of R^(ee) is, independently, selected from C₁₋₆ alkyl,C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ heteroalkyl, C₂₋₆heteroalkenyl, C₂₋₆heteroalkynyl, C₃₋₁₀ carbocyclyl, C₆₋₁₀ aryl, 3-10membered heterocyclyl, and 3-10 membered heteroaryl, wherein each alkyl,alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(u) groups;

each instance of R^(ff) is, independently, selected from hydrogen, C₁₋₆alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ heteroalkyl,C₂₋₆ heteroalkenyl, C₂₋₆heteroalkynyl, C₃₋₁₀ carbocyclyl, 3-10 memberedheterocyclyl, C₆₋₁₀ aryl and 5-10 membered heteroaryl, or two R^(ff)groups are joined to form a 3-14 membered heterocyclyl or 5-14 memberedheteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(gg)groups; and

each instance of R^(gg) is, independently, halogen, —CN, —NO₂, —N₃,—SO₂H, —SO₃H, —OH, —OC₁₋₆ alkyl, —ON(C₁₋₆alkyl)₂, —N(C₁₋₆ alkyl)₂,—N(C₁₋₆alkyl)₃ ⁴X⁻, —NH(C₁₋₆ alkyl)₂+X⁻, —NH₂(C₁₋₆ alkyl)⁺X⁻, —NH₃ ⁺X⁻,—N(OC₁₋₆ alkyl)(C₁₋₆ alkyl), —N(OH)(C₁₋₆ alkyl), —NH(OH), —SH, —SC₁₋₆alkyl, —SS(C₁₋₆ alkyl), —C(═O)(C₁₋₆alkyl), —CO₂H, —CO₂(C₁₋₆ alkyl),—OC(═O)(C₁₋₆ alkyl), —OCO₂(C₁₋₆alkyl), —C(═O)NH₂, —C(═O)N(C₁₋₆ alkyl)₂,—OC(═O)NH(C₁₋₆ alkyl), —NHC(═O)(C₁₋₆ alkyl), —N(C₁₋₆ alkyl)C(═O)(C₁₋₆alkyl), —NHCO₂(C₁₋₆ alkyl), —NHC(═O)N(C₁₋₆ alkyl)₂, —NHC(═O)NH(C₁₋₆alkyl), —NHC(═O)NH₂, —C(═NH)O(C₁₋₆alkyl), —OC(═NH)(C₁₋₆ alkyl),—OC(═NH)OC₁₋₆ alkyl, —C(═NH)N(C₁₋₆ alkyl)₂, —C(═NH)NH(C₁₋₆ alkyl),—C(═NH)NH₂, —OC(═NH)N(C₁₋₆ alkyl)₂, —OC(NH)NH(C₁₋₆alkyl), —OC(NH)NH₂,—NHC(NH)N(C₁₋₆ alkyl)₂, —NHC(═NH)NH₂, —NHSO₂(C₁₋₆ alkyl), —SO₂N(C₁₋₆alkyl)₂, —SO₂NH(C₁₋₆ alkyl), —SO₂NH₂, —SO₂C₁₋₆ alkyl, —SO₂OC₁₋₄ alkyl,—OSO₂C₁₋₆ alkyl, —SOC₁₋₆ alkyl, —Si(C₁₋₆ alkyl)₃, —OSi(C₁₋₆alkyl)₃-C(═S)N(C₁₋₆ alkyl)₂, C(═S)NH(C₁₋₆ alkyl), C(═S)NH₂, —C(═O)S(C₁₋₆alkyl), —C(═S)SC₁₋₆ alkyl, —SC(═S)SC₁₋₆ alkyl, —P(═O)₂(C₁₋₆ alkyl),—P(═O)(C₁₋₆alkyl)₂, —OP(═O)(C₁₋₆ alkyl)₂, —OP(═O)(OC₁₋₆alkyl)₂, C₂₋₆alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆heteroalkyl,C₂₋₆ heteroalkenyl, C₂₋₆heteroalkynyl, C₃₋₁₀ carbocyclyl, C₆₋₁₀ aryl,3-10 membered heterocyclyl, 5-10 membered heteroaryl; or two geminalR^(gg) substituents can be joined to form ═O or ═S; wherein X⁻ is acounterion.

As used herein, the term “halo” or “halogen” refers to fluorine (fluoro,—F), chlorine (chloro, —Cl), bromine (bromo, —Br), or iodine (iodo, —I).

As used herein, a “counterion” is a negatively charged group associatedwith a positively charged quarternary amine in order to maintainelectronic neutrality. Exemplary counterions include halide ions (e.g.,F⁻, Cl⁻, Br⁻, I⁻), NO₃ ⁻, ClO₄ ⁻, OH⁻, H₂PO₄ ⁻, HSO₄ ⁻, sulfonate ions(e.g., methanesulfonate, trifluoromethanesulfonate, p-toluenesulfonate,benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate,naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonicacid-2-sulfonate, and the like), and carboxylate ions (e.g., acetate,ethanoate, propanoate, benzoate, glycerate, lactate, tartrate,glycolate, and the like).

As used herein, the term “carbonyl” refers a group wherein the carbondirectly attached to the parent molecule is sp² hybridized, and issubstituted with an oxygen, nitrogen or sulfur atom, e.g., a groupselected from ketones (═C(═O)R^(aa)), carboxylic acids (═CO₂H),aldehydes (═CHO), esters (═CO₂R^(aa), —C(═O)SR^(aa), —C(═S)SR^(aa)),amides (═C(═O)N(R^(bb))₂, —C(═—O)NR^(bb)SO₂R^(aa), —C(═S)N(R^(bb))₂),and imines (═—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa)),—C(═NR^(bb))N(R^(bb))₂), wherein R^(aa) and R^(bb) are as definedherein.

As used herein, “azide” or “azido” refers to the group —N₃.

As used herein, the term “thiol” or “thio” refers to the group —SH. Theterm “substituted thiol” or “substituted thio,” by extension, refers toa thiol group wherein the sulfur atom directly attached to the parentmolecule is substituted with a group other than hydrogen, and includesgroups selected from —SR^(aa), —S═SR^(aa), —SC(═S)SR^(aa),—SC(═O)SR^(aa), —SC(═O)OR^(aa), and —SC(═O)R^(aa), wherein R^(aa) andR^(cc) are as defined herein.

As used herein, the term, “amino” or “amine” refers to the group —NH₂.The term “substituted” amino or amine, by extension, refers to amonosubstituted amino, a disubstituted amino, or a trisubstituted amino,as defined herein. In certain embodiments, the “substituted amino” is amonosubstituted amino or a disubstituted amino group.

As used herein, the term “monosubstituted amino” or “monosubstitutedamine” refers to an amino group wherein the nitrogen atom directlyattached to the parent molecule is substituted with one hydrogen and onegroup other than hydrogen, and includes groups selected from—NH(R^(bb)), —NHC(═O)R^(aa), —NHCO₂R^(aa), —NHC(═O)N(R^(bb))₂,—NHC(═NR^(bb))N(R^(bb))₂, —NHSO₂R^(aa), —NHP(═O)(OR^(cc))₂, and—NHP(═O)(NR^(bb))₂, wherein R^(aa), R^(bb) and R^(cc) are as definedherein, and wherein R^(bb) of the group —NH(R^(bb)) is not hydrogen.

As used herein, the term “disubstituted amino” or “disubstituted amine”refers to an amino group wherein the nitrogen atom directly attached tothe parent molecule is substituted with two groups other than hydrogen,and includes groups selected from —N(R^(bb))₂, —NR^(bb) C(═O)R^(aa),—NR^(bb)CO₂R^(aa), —NR^(bb)C(═O)N(R^(bb))₂,—NR^(bb)C(═NR^(bb))N(R^(bb))₂, —NR^(bb)SO₂R^(aa),—NR^(bb)P(═O)(OR^(cc))₂, and —NR^(bb)P(═O)(NR^(bb))₂, wherein R^(aa),R^(bb), and R^(cc) are as defined herein, with the proviso that thenitrogen atom directly attached to the parent molecule is notsubstituted with hydrogen.

As used herein, the term “trisubstituted amino” or “trisubstitutedamine” refers to an amino group wherein the nitrogen atom directlyattached to the parent molecule is substituted with three groups, andincludes groups selected from —N(R^(bb))₃ and —N(R^(bb))₃ ⁺X⁻, whereinR^(bb) and X⁻ are as defined herein.

As used herein, “biotin”, e.g., as an exemplary R^(1A) group, comprisesthe structure:

As used herein, a “ceramide”, e.g., as an exemplary R^(1A) group,comprises the structure:

wherein R′ is an optionally substituted C₆₋₃₀ alkyl (e.g., C₆, C₇, C₈,C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂,C₂₃, C₂₄, C₂₅, C₂₆, C₂₇, C₂₈, C₂₉, or C₃₀ alkyl), optionally substitutedC₆-C₃₀alkenyl (e.g., C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₆, C₁₇,C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, C₂₅, C₂₆, C₂₇, C₂₈, C₂₉, or C₃₀alkenyl), or optionally substituted C₆-C₃₀alkynyl (e.g., C₆, C₇, C₈, C₉,C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃,C₂₄, C₂₅, C₂₆, C₂₇, C₂₈, C₂₉, or C₃₀ alkynyl) group.

Nitrogen atoms can be substituted or unsubstituted as valency permits,and include primary, secondary, tertiary, and quarternary nitrogenatoms. Exemplary nitrogen atom substitutents include, but are notlimited to, hydrogen, —OH, —OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa),—C(═O)N(R^(cc))₂, —CO₂R^(aa), —SO₂R^(aa), —C(═NR^(bb))R^(aa),—C(═NR^(aa))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc),—SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(aa), —C(═S)SR^(aa),—P(═)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)₂N(R^(cc))₂, —P(═O)(NR^(aa))₂,C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₁₋₁₀heteroalkyl, C₂₋₁₀ heteroalkenyl, C₂₋₁₀ heteroalkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl, or two R^(cc) groups attached to an N atom are joined toform a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring,wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl isindependently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups, andwherein R^(aa), R^(bb), R^(cc) and R^(dd) are as defined above.

In certain embodiments, the substituent present on the nitrogen atom isan nitrogen protecting group (also referred to herein as an “aminoprotecting group”). Nitrogen protecting groups include, but are notlimited to, —OH, —OR^(aa), —N(R^(cc))₂, —C(═O)R^(aa), —C(═O)N(R^(cc))₂,—CO₂R^(aa), —SO₂R^(aa), —C(═NR^(cc))R^(aa), —C(═NR^(aa))OR^(aa),—C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc), —SO₂OR^(cc),—SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc), C₁₋₁₀ alkyl(e.g., aralkyl, heteroaralkyl), C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₁₋₁₀heteroalkyl, C₂₋₁₀ heteroalkenyl, C₂₋₁₀ heteroalkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl groups, wherein each alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl,and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5R^(dd) groups, and wherein R^(aa), R^(bb), R^(cc) and R^(dd) are asdefined herein. Nitrogen protecting groups are well known in the art andinclude those described in detail in Protecting Groups in OrganicSynthesis, T. W. Greene and P. G. M. Wuts. 3^(rd) edition, John Wiley &Sons, 1999, incorporated herein by reference.

For example, nitrogen protecting groups such as amide groups (e.g.,—C(═O)R^(aa)) include, but are not limited to, formamide, acetamide,chloroacetamide, trichloroacetamide, trifluoroacetamide,phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxyacylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide and o-(benzoyloxymethyl)benzamide.

Nitrogen protecting groups such as carbamate groups (e.g.,—C(═O)OR^(aa)) include, but are not limited to, methyl carbamate, ethylcarbamante, 9-fluorenylmethyl carbamate (Fmoc),9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethylcarbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1, l-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, r-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzylcarbamate.

Nitrogen protecting groups such as sulfonamide groups (e.g.,—S(═O)₂R^(aa)) include, but are not limited to, p-toluenesulfonamide(Ts), benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide(Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), 1-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Other nitrogen protecting groups include, but are not limited to,phenothiazinyl-(10)-acyl derivative, N′-p-toluenesulfonylaminoacylderivative, N′-phenylaminothioacyl derivative, N-benzoylphenylalanylderivative, N-acetylmethionine derivative,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate,N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).

In certain embodiments, the substituent present on an oxygen atom is anoxygen protecting group (also referred to herein as an “hydroxylprotecting group”). Oxygen protecting groups include, but are notlimited to, —R^(aa), —N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa),—CO₂R^(aa), —C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa),—C(═NR^(bb))N(R^(bb))₂, —S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃,—P(R^(cc))_(Z), —P(R^(cc))₃, —P(═O)₂R^(aa), —P(═O)(R^(aa))₂,—P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and —P(═O)(NR^(bb))₂, whereinR^(aa), R^(bb), and R^(cc) are as defined herein. Oxygen protectinggroups are well known in the art and include those described in detailin Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M.Wuts, 3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

Exemplary oxygen protecting groups include, but are not limited to,methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, α-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate,9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate(TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec),2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutylcarbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkylp-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzylcarbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzylcarbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate,4-ethoxy-1-naphthyl carbonate, methyl dithiocarbonate, 2-iodobenzoate,4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinate, (E)-2-methyl-2-butenoate,o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts).

These and other exemplary substituents are described in more detail inthe Detailed Description, Examples, and claims. The invention is notintended to be limited in any manner by the above exemplary listing ofsubstituents.

As used herein, the term “salt” refers to any and all salts.

The term “pharmaceutically acceptable salt” refers to those salts whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of humans and lower animals without unduetoxicity, irritation, allergic response and the like, and arecommensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, Berge et al.,describes pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptablesalts of the compounds of this invention include those derived fromsuitable inorganic and organic acids and bases. Examples ofpharmaceutically acceptable, nontoxic acid addition salts are salts ofan amino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, oxalic acid, maleic acid,tartaric acid, citric acid, succinic acid or malonic acid or by usingother methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Pharmaceutically acceptable salts derived from appropriatebases include alkali metal, alkaline earth metal, ammonium andN⁺(C₁₋₄alkyl)₄ salts. Representative alkali or alkaline earth metalsalts include sodium, lithium, potassium, calcium, magnesium, and thelike. Further pharmaceutically acceptable salts include, whenappropriate, nontoxic ammonium, quaternary ammonium, and amine cationsformed using counterions such as halide, hydroxide, carboxylate,sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the chemical structures of allyl-tailed Gb3, Gb4, Gb5,Globo H, and SSEA4.

FIG. 2 depicts glycosylation reactions combined with nucleotide sugarregeneration and synthesis results monitored by TLC. A: Combinedgalactosylation with UDP-Gal regeneration for synthesizing, e.g.,allyl-Gb3. B: Combined acetylgalactosamination with UDP-GalNAcregeneration for synthesizing, e.g., allyl-Gb4. C: Combinedgalactosylation with UDP-Gal regeneration for synthesizing, e.g.,allyl-Gb5. D: Combined fucosylation with GDP-Fuc regeneration forsynthesizing, e.g., allyl-Globo H. E: Combined sialylation withCMP-Neu5Ac regeneration for synthesizing, e.g., allyl-SSEA4.

FIG. 3 depicts the biosynthetic pathway of glycosphingolipids, involvingaddition of galactose residues, which can be calayzed by agalactosyltransferase coupled with the UDP-Gal regeneration processdescribed herein.

FIG. 4 depicts the enzymatic synthetic strategy in the manufacture ofGlobo H via the Lac→Gb3→Gb4→Gb5 pathway using a nucleotide sugarregeneration system.

FIG. 5 depicts the enzymatic synthetic strategy in the manufacture ofSSEA4 via the Lac→Gb3→Gb4→Gb5 pathway using a nucleotide sugarregeneration system.

FIG. 6 depicts the enzymatic synthetic strategy in the manufacture ofallyl-Globo H via the allyl-Lac→allyl-Gb3→allyl-Gb4→allyl-Gb5 pathwayusing a nucleotide sugar regeneration system.

FIG. 7 depicts the enzymatic synthetic strategy in the manufacture ofallyl-SSEA4 via the allyl-Lac→allyl-Gb3→allyl-Gb4→allyl-Gb5 pathwayusing a nucleotide sugar regeneration system.

FIG. 8 depicts the high purity obtained in the biosynthesis ofintermediates allyl-Gb3, allyl-Gb4, and allyl-Gb5.

FIG. 9 depicts the high purity obtained in the biosynthesis ofallyl-Globo H from allyl-Gb5 using unmodified and modified FutC.

FIG. 10 depicts the high purity obtained in the biosynthesis ofallyl-SSEA4 from allyl-Gb5 using JT-FAJ-16.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are newly developed nucleotide sugar regenerationprocesses and their uses in adding sugar residues to suitable acceptorsvia the action of a suitable glycosyltransferase. These approaches allowchain reactions for synthesizing glycosylated molecules, such asoligosaccharides (e.g., Gb3, Gb4, Gb5, Globo H, and SSEA4) without theneed to purify intermediates, resulting in unexpectedly rapid productionof the glycosylated products with unexpectedly high yields. In addition,the synthesis methods described herein can be used for large scaleproduction of desired oligosaccharides and glycoconjugates.

UDP-Gal Regeneration System and its Use in Galactosylation

The UDP-Gal regeneration system is exemplified in FIG. 2A, involving theenzymes listed in Table 1 below:

TABLE 1 Enzymes Used in UDP-Gal Regeneration System Enzyme ActivityExamples Galactokinase (GalK) Catalyzes the phosphorylation of E. coli(e.g., GenBank accession alpha-D-galactose to produce no. AP012306.1galactose-1-phosphate (Gal-1-P) H. sapiens (e.g., GenBank in thepresence of ATP accession no. NP_000145) M. hydrothermalis (e.g.,GenBank accession no. YP_004368991) S. sputigena (e.g., GenBankaccession no. AEC00832) H. hydrossis (e.g., GenBank accession no.YP_004451189) UDP-sugar Catalyzes the conversion of Gal- A. thaliana(e.g., GenBank pyrophosphorylase 1-P to UDP-Gal in the presenceaccession no. AF360236.1 (USP) of UTP L. major (e.g., GenBank accessionno. ABY79093) T. cruzi (e.g., GenBank accession no. ADD10758) L.donovani (e.g., GenBank accession no. XP_00385998) G. max (e.g., GenBankaccession no. NP_001237434) Pyruvate kinase Catalyzes the transfer of aE. coli (e.g., GenBank accession (PykF) phosphate group from no.U00096.2) phosphoenolpyruvate (PEP) to N. hamburgensis (e.g., GenBankADP, producing pyruvate and accession no. YP_576506) ATP or UTP R.palustris (e.g., GenBank accession no. YP_7830161) M. ruestringensis(e.g., GenBank accession no. YP_004787669) H. hydrossis (e.g., GenBankaccession no. YP_004450514) S. coccoides (e.g., GenBank accession no.YP_00441096) Pyrophosphatase Acid anhydride hydrolase that E. coli(e.g., GenBank accession (PPA) (Optional) acts upon diphosphate bondsno. U00096.2 G. theta (e.g., GenBank accession no. CAI77906) C.butyricum (e.g., GenBank accession no. ZP_04525837) L. plantarum (e.g.,GenBank accession no. EFK28054) L. suebicus (e.g., GenBan accession no.ZP_09451344)

The enzymes to be used in the UDP-Gal regeneration system describedherein can be a wild-type enzyme. As used herein, a wild-type enzyme isa naturally occurring enzyme found in a suitable species. In someexamples, the GalK, USP, PykF, and PPA enzymes can be from E. coli, A.thaliana, E. coli, and E. coli, respectively. Examples of the enzymesfrom these species are listed in Table 1 above. Others can be readilyidentified by those skilled in the art, e.g., search a publiclyavailable gene database, such as GenBank. In other examples, theseenzymes are homologs of those from the just-noted species, which arewithin the knowledge of those skilled in the art. For example, suchhomologs can be identified by searching GenBank using the amino acidsequence or the coding nucleotide sequence of an exemplary enzyme as asearch query.

Alternatively, the enzymes involved in the UDP-Gal regeneration systemcan be a functional variant of a wild-type counterpart. As used herein,a functional variant of a wild-type enzyme possesses the same enzymaticactivity as the wild-part counterpart and typically shares a high aminoacid sequence homology, e.g., at least 80%, 85%, 90%, 95, or 98%identical to the amino acid sequence of the wild-type counterpart. The“percent identity” of two amino acid sequences is determined using thealgorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68,1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST andXBLAST programs (version 2.0) of Altschul, et al J. Mol. Biol.215:403-10, 1990. BLAST protein searches can be performed with theXBLAST program, score=50, wordlength=3 to obtain amino acid sequenceshomologous to the protein molecules of interest. Where gaps existbetween two sequences, Gapped BLAST can be utilized as described inAltschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used.

A functional variant can have various mutations, including addition,deletion, or substitution of one or more amino acid residues. Such avariant often contain mutations in regions that are not essential to theenzymatic activity of the wild-type enzyme and may contain no mutationsin functional domains or contain only conservative amino acidsubstitutions. The skilled artisan will realize that conservative aminoacid substitutions may be made in lipoic acid ligase mutants to providefunctionally equivalent variants, i.e., the variants retain thefunctional capabilities of the particular lipoic acid ligase mutant. Asused herein, a “conservative amino acid substitution” refers to an aminoacid substitution that does not alter the relative charge or sizecharacteristics of the protein in which the amino acid substitution ismade. Variants can be prepared according to methods for alteringpolypeptide sequence known to one of ordinary skill in the art such asare found in references which compile such methods, e.g. MolecularCloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. Conservative substitutions of aminoacids include substitutions made amongst amino acids within thefollowing groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G;(e) S, T; (f) Q, N; and (g) E, D.

Any of the enzymes involved in the UDP-Gal regeneration system can beprepared via routine technology. In one example, the enzyme is isolatedform a natural source. In other examples, the enzyme is prepared byroutine recombinant technology. When necessary, the coding sequence of atarget enzyme can be subjected to coden optimization based on the hostcell used for producing the enzyme. For example, when E. coli cells areused as the host for producing an enzyme via recombinant technology, thegene encoding that enzyme can be modified such that it contains codonscommonly used in E. coli.

As illustrated in FIG. 2A, the UDP-Gal regeneration system can be usedin conjunction with a galactosylation reaction via the activity of agalactosyltransferase to add a galactose residue to a suitablesubstrate. Examples of galactosyltransferases are listed in Table 2below:

TABLE 2 Galactosyltransferases Galactosyltransferase Enzymatic ActivityExamples Beta-1,4-Galactosyltransferase Catalyzes the transfer of Homosapiens [e.g., GI: (B4GALT), including isoforms galactose from UDP-Galto 903740] 1-7 a suitable acceptor, such as Rattus norvegicus [e.g., GI:(Beta-1,4-galactosyltransferase a glycoprotein or glycolipid 3258653]1-7) acceptor having a terminal Zobellia galactanivorans [e.g.,2-acetamido-2-deoxy-D- GI: 340619721] glucosyl-group, in an Clostridiumperfringens [e.g., beta1,4-linkage GI: 18309463]Beta-1,3-Galactosyltransferase Catalyzes the transfer of Culexquinquefasciatus [e.g., (B3GALNT) galactose from UDP-Gal to GI:167873909] a suitable acceptor, such as Zea mays [e.g., GI: 195643406] aglycoprotein or glycolipid Brachyspira pilosicoli [e.g., GI: acceptorhaving a terminal 300871377] 2-acetamido-2-deoxy-D- Enterococcus faecium[e.g., GI: glucosyl-group, or a 257822935] GalNAc residue, in an LgtD,from, e.g., Haemophilus beta1,3-linkage influenza [L42023.1] Alpha-1,4-Catalyzes the transfer of a Homo sapiens [e.g., Galactosyltransferasegalactose from UDP-Gal to GI: 55956926] (A4GALT) a suitable acceptorsuch as a Mustela putorius furo [e.g., GI: e.g.: glycoprotein or aglycolipid 355666115] Lactosylceramide 4-alpha- having, e.g., a terminalMus musculus [e.g., GI: galactosyltransferase galactose residue or a51921295] GlcNAc residue in an alpha Rattus norvegicus [e.g., GI:1,4-linkage 67677925] LgtC from, e.g., Neisseria meningitides [e.g.,AF355193.1] Alpha-1,3- Catalyzes the transfer of a Mus musculus [e.g.,Galactosyltransferase galactose from UDP-Gal to GI: 224922807] (A3GALT)a suitable acceptor such as a Mustela putorius furo [e.g., GI: e.g.:glycoprotein or a glycolipid 355690122] Alpha-1,3- having, e.g., aterminal Cebus paella [e.g., GI: Galactosyltransferase 1 galactoseresidue or a 19698748] Alpha-1,3- GlcNAc residue in an alpha Rattusnorvegicus [e.g., GI: Galactosyltransferase 2 1,3-linkage 28625949]

Both wild-type galactosyltransferases and functional variants, asdescribed above, are within the scope of this description. Suchglycosyltransferases can be prepared via any routine method.

The combination of the UDP-Gal regeneration system and one or moregalactosyltransferases can be used for adding a galactose residue to asuitable substrate (an acceptor) with high yields. Substrates forgalactosyltransferase, e.g., described in Table 2 above, are well knownto those skilled in the art. Preferably, the substrate has a terminalsugar residue (e.g., Gal, GalNAc, or GlcNAc) to which the galactoseresidue can be added. In some examples, the substrate is apolysaccharide (having >50 monosaccharide units), an oligosaccharide(having 2-50 monosaccharide units), a glycoprotein or glycopeptide, or aglycolipid. The type of a galactosyltransferase to be used in thegalactosylation methods descried herein depends on the end product ofinterest and the substrate for synthesizing the end product, which iswell within the knowledge of a skilled artisan. The combined UDP-Galregeneration system/galactosyltransferase approach described herein canbe used to synthesize glycosphingolipids. Examples are illustrated inFIG. 3.

In other examples, the combined UDP-Gal generationsystem/galactosyltransferase approach can be used for synthesizingGlobo-series oligosaccharides, such as synthesis of Gb3 from lactose orsynthesis of Gb5 from Gb4. FIGS. 2A and 2C. See also descriptions below.

UDP-GalNAc Regeneration System and its Use in N-Acetylgalactosamination

A UDP-GalNAc regeneration system can be co-used with anN-acetylgalactosaminyltransferase (GalNAcT), such as abeta1,3-N-acetylgalactosaminyltransferase, for addition of a GalNAcresidue onto a suitable acceptor.

Enzymes involved in an exemplary UDP-GalNAc regeneration system areshown in Table 3 below:

TABLE 3 Enzymes Used in UDP-GalNAc Regeneration System Enzyme ActivityExamples N-Acetylhexosamine 1- Acts by a sequential two NahK from B.longum (e.g., Kinase (GalNAcK) substrates-two products GenBank accessionno. mechanism to convert ATP and CP000246.1 N-acetylhexosamine into ADPB. breve (e.g., GenBank and N-acetyl-alpha-D- accession no. ZP_06596651)hexosamine 1-phosphate. A. haemolyticum (e.g., GenBank accession no.YP_003696399 B. bifidum (e.g., GenBank accession no. YP_003938776)N-acetylglucosamine 1- Catalyzes the conversion of GlmU from E. coli(e.g., phosphate UTP and N-acetyl-alpha-D- GenBank accession no.uridylyltransferase (GlmU) glucosamine 1-phosphate into U00096.2diphosphate and UDP-N- A. thaliana (e.g., GenBank acetyl-D-glucosamineaccession no. AEE31311) G. bemidjiensis (e.g., GenBank accession no.ACH37122) H. pylori (e.g., GenBank accession no. YP_003728906) Pyruvatekinase (PykF) Catalyzes the transfer of a E. coli (e.g., GenBankphosphate group from accession no. U00096.2) phosphoenolpyruvate (PEP)to N. hamburgensis (e.g., ADP, producing pyruvate and GenBank accessionno. ATP or UTP YP_576506) R. palustris (e.g., GenBank accession no.YP_7830161) M. ruestringensis (e.g., GenBank accession no. YP_004787669)H. hydrossis (e.g., GenBank accession no. YP_004450514) S. coccoides(e.g., GenBank accession no. YP_00441096) Pyrophosphatase (PPA) Acidanhydride hydrolase that E. coli (e.g., GenBank (Optional) acts upondiphosphate bonds accession no. U00096.2 G. theta (e.g., GenBankaccession no. CAI77906) C. butyricum (e.g., GenBank accession no.ZP_04525837) L. plantarum (e.g., GenBank accession no. EFK28054) L.suebicus (e.g., GenBan accession no. ZP_09451344)

N-acetylgalactosaminyltransferase (e.g., beta-1,3-GalNAcT orbeta-1,4-GalNAcT) is an enzyme that catalyzes the reaction in which aGAlNAc residue is added onto a suitable acceptor, such as a peptide oran oligosaccharide. Examples include LgtD from H. influenza, (GenBankaccession no. L42023.1. Other examples include, but are not limited to,LgtD of B. garinii (e.g., GenBank accession no. AEW68905), LgtD of N.lactamica (e.g., GenBank accession no. AAN08512), and LgtD of R. felis(e.g., GenBank accession no. YP_246702).

Any of the enzymes used in the combined UDP-GalNAc regenerationsystem/GalNAcT approach can be either a wild-type enzyme or a functionalvariant thereof, as described herein. Any conventional method can beused for preparing such enzyme. In one example, this approach is appliedfor synthesizing Gb4 from Gb3. See, e.g., FIG. 2B.

GDP-Fuc Regeneration System and its Use in Fucosylation

An GDP-Fuc regeneration system can be co-used with a fucosyltransferase(e.g., an alpha1,2-fucosyltransferase, an alpha1,3-fucosyltransferase,or an alpha2,6-fucosyltransferase) to add a fucose residue to a suitableacceptor, such as an oligosaccharide, which can be conjugated to anothermolecule such as a lipid or a polypeptide.

Enzymes involved in an exemplary GDP-Fuc regeneration system are shownin Table 4 below:

TABLE 4 Enzymes Used in GDP-Fuc Regeneration System Enzyme ActivityExamples L-fucokinase/GDP-fucose A biofunctional enzyme that B. fragilis(e.g., GenBank pyrophosphorylase (FKP) generates Fuc-1-P and GDP-accession no. CR626927.1 Fuc from fucose and ATP H. sapiens (e.g.,GenBank accession no. NP_003829) R. norvegicus (e.g., GenBank accessionno. NP_955788) Pyruvate kinase (PykF) Catalyzes the transfer of a E.coli (e.g., GenBank accession phosphate group from no. U00096.2)phosphoenolpyruvate (PEP) to N. hamburgensis (e.g., GenBank ADP,producing pyruvate and accession no. YP_576506) ATP or UTP R. palustris(e.g., GenBank accession no. YP_7830161) M. ruestringensis (e.g.,GenBank accession no. YP_004787669) H. hydrossis (e.g., GenBankaccession no. YP_004450514) S. coccoides (e.g., GenBank accession no.YP_00441096) Pyrophosphatase (PPA) Acid anhydride hydrolase that E. coli(e.g., GenBank accession (Optional) acts upon diphosphate bonds no.U00096.2 G. theta (e.g., GenBank accession no. CAI77906) C. butyricum(e.g., GenBank accession no. ZP_04525837) L. plantarum (e.g., GenBankaccession no. EFK28054) L. suebicus (e.g., GenBan accession no.ZP_09451344)

A fucosyltransferase transfers an L-fucose sugar from a GDP-fucose(guanosine diphosphate-fucose) donor substrate to an acceptor substrate,which can be another sugar. Fucosyltransferase can add the fucoseresidue to a core GlcNAc (N-acetylglucosamine) sugar as in the case ofN-linked glycosylation, or to a protein, as in the case of O-linkedglycosylation. Fucosyltransferases include alpha1,3-fucosyltransferase,alpha1,2-fucosyltransferase, and alpha1,6-fucosyltransferase. Examplesinclude alpha1,2-fucosyltransferase from E. coli (e.g., GenBankaccession no. U00096.2), alpha 1,3-fucosyltransferase from B. fragilis(e.g., GenBank accession no. YP_213404) and from X. laevis (e.g.,GenBank accession no. NP_001083664), alpha 1,6-fucosyltransferase fromX. Any of the enzymes used in the combined GDP-Fuc regenerationsystem/FucT approach can be either a wild-type enzyme or a functionalvariant thereof, as described herein. Any conventional method can beused for preparing such enzyme. In one example, this approach is appliedfor synthesizing Gb4 from Gb3. See, e.g., FIG. 2D.

CMP-Neu5Ac Regeneration System and its Use in Sialylation

An CMP-Neu5Ac regeneration system can be coupled with asialyltransferase, such as an alpha 2,3-sialyltransferase, to add asialic acid residue (Neu5Ac) to a suitable acceptor substrate, such asan oligosaccharide.

Enzymes involved in an exemplary CMP-Neu5Ac regeneration system areshown in Table 5 below:

TABLE 5 Enzymes Used in CMP-Neu5Ac Regeneration System Enzyme ActivityExamples Cytidine monophosphate Catalyzes phosphorylation of E. coli(e.g., GenBank accession kinase (CMK) CMP to produce CDP no. U00096.2 B.amyloliquefaciens (e.g., GenBank accession no. ABS74466) M. leprae(e.g., GenBank accession no. CAB08279) M. avium (e.g., GenBank accessionno. AAS03731) B. garinii (e.g., GenBank accession no. AEW68468)CMP-sialic acid Catalyzes the synthesis of P. multocida (e.g., GenBanksynthetase (Css) CMP sialic acid from CTP accession no. AE004439.1 andsialic acid. N. meningitidis (e.g., GenBank accession no. AAB60780) O.mykiss (e.g., GenBank accession no. BAB47150) I. ioihiensis (e.g.,GenBank accession no. AAV81361) C. jejuni (e.g., GenBank accession no.ABI32334) Pyruvate kinase (PykF) Catalyzes the transfer of a E. coli(e.g., GenBank accession phosphate group from no. U00096.2)phosphoenolpyruvate (PEP) to N. hamburgensis (e.g., GenBank ADP,producing pyruvate and accession no. YP_576506) ATP or UTP R. palustris(e.g., GenBank accession no. YP_7830161) M. ruestringensis (e.g.,GenBank accession no. YP_004787669) H. hydrossis (e.g., GenBankaccession no. YP_004450514) S. coccoides (e.g., GenBank accession no.YP_00441096) Pyrophosphatase (PPA) Acid anhydride hydrolase that E. coli(e.g., GenBank accession (Optional) acts upon diphosphate bonds no.U00096.2 G. theta (e.g., GenBank accession no. CAI77906) C. butyricum(e.g., GenBank accession no. ZP_04525837) L. plantarum (e.g., GenBankaccession no. EFK28054) L. suebicus (e.g., GenBan accession no.ZP_09451344)

Sialyltransferases are enzymes that transfer sialic acid to nascentoligosaccharide. This family of enzymes adds sialic acid to the terminalportions of sialylated glycolipids (gangliosides) or to the N- orO-linked sugar chains of glycoproteins. There are about twenty differentsialyltransferases, including sialyltransferases that add sialic acidwith an alpha 2,3 linkage to galactose (e.g., alpha2,3-sialyltransferase), and sialyltransferases that add sialic acid withan alpha 2,6 linkage to galactose or N-acetylgalactosamine (e.g., alpha2,6-sialyltransferase). Examples include alpha 2,3-sialyltransferasefrom, e.g., M. bacteria (GenBank accession no. AB308042.1), M. musculus(e.g., GenBank accession no. BAA06068), or P. multocida (e.g., GenBankaccession no. AET17056); and alpha 2,6-sialyltransferase from, e.g., B.taurus (e.g., GenBank accession no. NP_001008668), C. griseus (e.g.,GenBank accession no. NP_001233744), or R. norvegicus (e.g., GenBankaccession no. AAC42086).

Any of the enzymes used in the combined CMP-Neu5Ac regenerationsystem/sialyltransferase approach can be either a wild-type enzyme or afunctional variant thereof, as described herein. Any conventional methodcan be used for preparing such enzyme. In one example, this approach isapplied for synthesizing Gb4 from Gb3. FIG. 2E.

Synthesis of Globo-Series Oligosaccharides

The above-described combined approaches involving UDP-Galregeneration/galactosyltransferase, UDP-GalNAc regeneration/GalNAcT,GDP-Fuc regeneration/fucosyltransferase, and CMP-Neu5Acregeneration/sialyltransferase can be applied, either independently, orin combination, to synthesize Globo-series oligosaccharides, includingGb3. Gb4, Gb5, Globo H (fucosyl-Gb5), and SSEA4 (sialyl-Gb5). Asdiscussed in greater detail below, all of these Globo-seriesoligosaccharides can be either substituted or unsubstituted.

Step S-1

The first step in the biosynthetic approach (S-1) involves enzymaticconversion of a compound of Formula (I), or salt thereof, to a compoundof Formula (II), or salt thereof:

wherein R^(1A) is hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, or an oxygen protecting group;and each instance of R², R^(3A), R^(5A), R^(2B), R^(3B), R^(5B), R^(2C),R^(4C), and R^(5C) is independently hydrogen, substituted orunsubstituted C₁₋₆ alkyl, or an oxygen protecting group.

Thus, in one aspect, provided is a method of enzymatically synthesizinga compound of Formula (II), or salt thereof, from a compound of Formula(I), or salt thereof, comprising converting a compound of Formula (I) toa compound of Formula (H), or salt thereof, in the presence of uridinediphosphate-Gal (UDP-Gal) and an alpha-1,4 galactosyltransferase, andregenerating UDP-Gal from galactose in the presence of the set ofenzymes listed in Table 1 above. See, e.g., FIG. 2A. To perform thisenzymatic reaction, necessary components, such as galactose,galactosyltransferase, the set of UDP-Gal regeneration enzymes, ATP,UTP, and others (e.g., Mg⁺⁺), can be mix to form a reaction mixture,which can be incubated under suitable conditions allowing production ofFormula (II) compounds. Such conditions are well known to those skilledin the art. See also Examples below.

The R^(1A) group can serve as a functional group allowing conjugation ofthe Globo-series oligosaccharides to another molecule, such as a proteinor a lipid. Alternative, it can serve as a protecting group.

In certain embodiments, R^(1A) is hydrogen.

In other embodiments, R^(1A) is substituted or unsubstituted alkyl,e.g., substituted or unsubstituted C₁₋₆alkyl, substituted orunsubstituted C₂₋₆alkyl, substituted or unsubstituted C₃₋₆alkyl,substituted or unsubstituted C₄₋₆alkyl, substituted or unsubstitutedC₅₋₆alkyl, substituted or unsubstituted C₂₋₅alkyl, substituted orunsubstituted C₂₋₄alkyl, substituted or unsubstituted C₂₋₃alkyl,substituted or unsubstituted C₁alkyl, substituted or unsubstitutedC₂alkyl, substituted or unsubstituted C₃alkyl, substituted orunsubstituted C₄alkyl, substituted or unsubstituted C₅alkyl, orsubstituted or unsubstituted C₆alkyl. Biotin and a ceramide, as definedherein, are encompassed by substituted alkyl. In certain embodiments,R^(1A) is an unsubstituted alkyl, e.g., in certain embodiments, R^(1A)is methyl, ethyl, propyl, isopropyl, sec-butyl, iso-butyl, ortert-butyl. Alternatively, in certain embodiments, R^(1A) is asubstituted alkyl. In certain embodiments, R^(1A) is alkyl which isfurther substituted with a substituted or unsubstituted thio,substituted or unsubstituted amino, carbonyl (e.g., carboxylic acid),azido, alkenyl (e.g., allyl), alkynyl (e.g., propargyl), biotin, or aceramide group. In certain embodiments, such substituents aresubstituted at the terminal position (last carbon atom) of the alkylgroup. In certain embodiments, R^(1A) is alkyl substituted with one ormore amino (—NH₂) groups. In certain embodiments, R^(1A) is alkylsubstituted at the terminal position (last carbon atom) with an amino(═NH₂) group. In certain embodiments, R^(1A) is —(CH₂)_(n)—NH₂ wherein nis 1, 2, 3, 4, 5, or 6. In certain embodiments, R^(1A) is 5-pentylamino(—(CH₂)₅—NH₂).

In certain embodiments, R^(1A) is substituted or unsubstituted alkenyl,e.g., substituted or unsubstituted C₂₋₆alkenyl, substituted orunsubstituted C₃₋₆alkenyl, substituted or unsubstituted C₄₋₆alkenyl,substituted or unsubstituted C₅₋₆alkenyl, substituted or unsubstitutedC₂₋₅alkenyl, substituted or unsubstituted C₂₋₄alkenyl, substituted orunsubstituted C₂₋₃alkenyl, substituted or unsubstituted C₂alkenyl,substituted or unsubstituted C₃alkenyl, substituted or unsubstitutedC₄alkenyl, substituted or unsubstituted C₅alkenyl, or substituted orunsubstituted C₆alkenyl. In certain embodiments, R^(1A) is—(CH₂)_(m)—CH═CH₂, wherein n is 1, 2, or 3. In certain embodiments,R^(1A) is allyl (—CH₂CH═CH₂). In certain embodiments, R^(1A) is alkenylwhich is further substituted with a substituted or unsubstituted thio,substituted or unsubstituted amino, carbonyl (e.g., carboxylic acid),azido, alkenyl (e.g., allyl), alkynyl (e.g., propargyl), biotin, or aceramide group. In certain embodiments, such substituents aresubstituted at the terminal position (last carbon atom) of the alkenylgroup

In certain embodiments, R^(1A) is substituted or unsubstituted alkynyl,e.g., substituted or unsubstituted C₂₋₆alkynyl, substituted orunsubstituted C₃₋₆alkynyl, substituted or unsubstituted C₄₋₆alkynyl,substituted or unsubstituted C₅₋₆alkynyl, substituted or unsubstitutedC₂₋₅alkynyl, substituted or unsubstituted C₂₋₄alkynyl, substituted orunsubstituted C₂₋₃alkynyl, substituted or unsubstituted C₂alkynyl,substituted or unsubstituted C₃alkynyl, substituted or unsubstitutedC₄alkynyl, substituted or unsubstituted C₅alkynyl, or substituted orunsubstituted C₆alkynyl. In certain embodiments, R^(1A) is alkynyl whichis further substituted with a substituted or unsubstituted thio,substituted or unsubstituted amino, carbonyl (e.g., carboxylic acid),azido, alkenyl (e.g., allyl), alkynyl (e.g., propargyl), biotin, or aceramide group. In certain embodiments, such substituents aresubstituted at the terminal position (last carbon atom) of the alkynylgroup.

In certain embodiments, R^(1A) is substituted or unsubstitutedheterocyclyl, e.g., substituted or unsubstituted 5- to 8-memberedheterocyclyl, substituted or unsubstituted 5- to 7-memberedheterocyclyl, substituted or unsubstituted 5- to 6-memberedheterocyclyl, substituted or unsubstituted 5-membered heterocyclyl,substituted or unsubstituted 6-membered heterocyclyl, substituted orunsubstituted 7-membered heterocyclyl, or substituted or unsubstituted8-membered heterocyclyl. In certain embodiments, R^(1A) is heterocyclylwhich is further substituted with a substituted or unsubstituted thio,substituted or unsubstituted amino, carbonyl (e.g., carboxylic acid),azido, alkenyl (e.g., allyl), alkynyl (e.g., propargyl), biotin, or aceramide group.

In certain embodiments, R^(1A) is substituted or unsubstitutedcarbocyclyl, e.g., substituted or unsubstituted C₃₋₆ carbocyclyl,substituted or unsubstituted C₃₋₅ carbocyclyl, substituted orunsubstituted C₃₋₄ carbocyclyl, substituted or unsubstituted C₃carbocyclyl, substituted or unsubstituted C₄ carbocyclyl, substituted orunsubstituted C₅ carbocyclyl, or substituted or unsubstituted C₆carbocyclyl. In certain embodiments, R^(1A) is carbocyclyl which isfurther substituted with a substituted or unsubstituted thio,substituted or unsubstituted amino, carbonyl (e.g., carboxylic acid),azido, alkenyl (e.g., allyl), alkynyl (e.g., propargyl), biotin, or aceramide group.

In certain embodiments, R^(1A) is substituted or unsubstituted aryl,e.g., substituted or unsubstituted C₆ aryl (phenyl) or substituted orunsubstituted C₁₀ aryl (naphthyl). In certain embodiments, R^(1A) isaryl which is further substituted with a substituted or unsubstitutedthio, substituted or unsubstituted amino, carbonyl (e.g., carboxylicacid), azido, alkenyl (e.g., allyl), alkynyl (e.g., propargyl), biotin,or a ceramide group.

In certain embodiments, R^(1A) is substituted or unsubstitutedheteroaryl, e.g., substituted or unsubstituted 5-membered heteroaryl orsubstituted or unsubstituted 6-membered heteroaryl. In certainembodiments, R^(1A) is heteroaryl which is further substituted with asubstituted or unsubstituted thio, substituted or unsubstituted amino,carbonyl (e.g., carboxylic acid), azido, alkenyl (e.g., allyl), alkynyl(e.g., propargyl), biotin, or a ceramide group.

In certain embodiments, R^(1A) is hydrogen, allyl, substituted alkyl,biotin, or a ceramide.

It is further contemplated herein that R^(1A) can be a mixture of any ofthe above recited non-hydrogen groups, e.g., substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, substituted or unsubstituted carbocyclyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, to provide a linker groupcomprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different combinations ofgroups. As a non-limiting example, R^(1A) may be a linker groupcomprising alkyl and aryl combination of groups, e.g., such asalkyl-aryl-alkyl, and which may optionally be further substituted at anyposition on the linker group (e.g., the terminal position) with asubstituted or unsubstituted thio, substituted or unsubstituted amino,carbonyl (e.g., carboxylic acid), azido, alkenyl (e.g., allyl), alkynyl(e.g., propargyl), biotin, or a ceramide group.

In certain embodiments, R^(1A) is an oxygen protecting group, as definedherein.

In certain embodiments, R^(2A) is hydrogen. In certain embodiments,R^(2A) is substituted or unsubstituted C₁₋₆ alkyl, e.g., substituted orunsubstituted C₁alkyl, substituted or unsubstituted C₂alkyl, substitutedor unsubstituted C₃alkyl, substituted or unsubstituted C₄alkyl,substituted or unsubstituted C₅alkyl, or substituted or unsubstitutedC₆alkyl. In certain embodiments, R^(2A) is an oxygen protecting group.

In certain embodiments, R^(3A) is hydrogen. In certain embodiments,R^(3A) is substituted or unsubstituted C₁₋₆ alkyl, e.g., substituted orunsubstituted C₁alkyl, substituted or unsubstituted C₂alkyl, substitutedor unsubstituted C₃alkyl, substituted or unsubstituted C4alkyl,substituted or unsubstituted C₅alkyl, or substituted or unsubstitutedC₆alkyl. In certain embodiments, R^(3A) is an oxygen protecting group.

In certain embodiments, R^(5A) is hydrogen. In certain embodiments,R^(5A) is substituted or unsubstituted C₁₋₆ alkyl, e.g., substituted orunsubstituted C₁alkyl, substituted or unsubstituted C₂alkyl, substitutedor unsubstituted C₃alkyl, substituted or unsubstituted C₄alkyl,substituted or unsubstituted C₅alkyl, or substituted or unsubstitutedC₆alkyl. In certain embodiments, R^(SA) is an oxygen protecting group.

In certain embodiments, R^(2B) is hydrogen. In certain embodiments,R^(2B) is substituted or unsubstituted C₁₋₆alkyl, e.g., substituted orunsubstituted C₁alkyl, substituted or unsubstituted C₂alkyl, substitutedor unsubstituted C₃alkyl, substituted or unsubstituted C₄alkyl,substituted or unsubstituted C₅alkyl, or substituted or unsubstitutedC₆alkyl. In certain embodiments, R^(2B) is an oxygen protecting group.

In certain embodiments, R^(3B) is hydrogen. In certain embodiments,R^(3B) is substituted or unsubstituted C₁₋₆ alkyl, e.g., substituted orunsubstituted C₁alkyl, substituted or unsubstituted C₂alkyl, substitutedor unsubstituted C₃alkyl, substituted or unsubstituted C₄alkyl,substituted or unsubstituted C₅alkyl, or substituted or unsubstitutedC₆alkyl. In certain embodiments, R^(3B) is an oxygen protecting group.

In certain embodiments, R^(5B) is hydrogen. In certain embodiments,R^(5B) is substituted or unsubstituted C₁₋₆ alkyl, e.g., substituted orunsubstituted C₁alkyl, substituted or unsubstituted C₂alkyl, substitutedor unsubstituted C₃alkyl, substituted or unsubstituted C₄alkyl,substituted or unsubstituted C₅alkyl, or substituted or unsubstitutedC₆alkyl. In certain embodiments, R^(5B) is an oxygen protecting group.

In certain embodiments, R^(2C) is hydrogen. In certain embodiments,R^(2C) is substituted or unsubstituted C₁₋₆alkyl, e.g., substituted orunsubstituted C₁alkyl, substituted or unsubstituted C₂alkyl, substitutedor unsubstituted C₃alkyl, substituted or unsubstituted C₄alkyl,substituted or unsubstituted C₅alkyl, or substituted or unsubstitutedC₆alkyl. In certain embodiments, R^(2C) is an oxygen protecting group.

In certain embodiments, R^(4C) is hydrogen. In certain embodiments,R^(4C) is substituted or unsubstituted C₁₋₆ alkyl, e.g., substituted orunsubstituted C₁alkyl, substituted or unsubstituted C₂alkyl, substitutedor unsubstituted C₃alkyl, substituted or unsubstituted C₄alkyl,substituted or unsubstituted C₅alkyl, or substituted or unsubstitutedC₆alkyl. In certain embodiments, R^(2C) is an oxygen protecting group.

In certain embodiments, R^(4C) is hydrogen. In certain embodiments,R^(4C) is substituted or unsubstituted C₁₋₆ alkyl, e.g., substituted orunsubstituted C₁alkyl, substituted or unsubstituted C₂alkyl, substitutedor unsubstituted C₃alkyl, substituted or unsubstituted C₄alkyl,substituted or unsubstituted C₅alkyl, or substituted or unsubstitutedC₆alkyl. In certain embodiments, R^(4C) is an oxygen protecting group.

In certain embodiments, each instance of R^(2A), R^(3A), R^(5A), R^(2B),R^(3B), R^(5B), R^(2C), R^(4C), and R^(5C) is independently hydrogen. Incertain embodiments, R^(1A) is substituted or unsubstituted alkenyl, andeach instance of R^(2A), R^(3A), R^(5A), R^(2B), R^(3B), R^(5B), R^(2C),R^(4C), and R^(5C) is independently hydrogen. In certain embodiments,R^(1A) is substituted or unsubstituted alkyl, and each instance ofR^(2A), R^(3A), R^(5A), R^(2B), R^(3B), R^(5B), R^(2C), R^(4C), andR^(5C) is independently hydrogen.

Exemplary compounds of Formula (I) include, but are not limited to, andsalts thereof.

Exemplary compounds of Formula (II) include, but are not limited to,

and salts thereof.

Step S-2

The second step in the biosynthetic approach (S-2) involves enzymaticconversion of a compound of Formula (II), or salt thereof, to a compoundof Formula (III), or salt thereof:

wherein R^(1A), R^(2A), R^(3A), R^(5A), R^(2B), R^(3B), R^(5B), R^(2C),R^(4C), and R^(5C) are as defined herein; and each instance of R^(4D)and R^(5D) is independently hydrogen, substituted or unsubstituted C₁₋₆alkyl, or an oxygen protecting group.

In certain embodiments, R^(2D) is hydrogen. In certain embodiments,R^(2D) is substituted or unsubstituted C₁₋₆ alkyl, e.g., substituted orunsubstituted C₁alkyl, substituted or unsubstituted C₂alkyl, substitutedor unsubstituted C₃alkyl, substituted or unsubstituted C₄alkyl,substituted or unsubstituted C₅alkyl, or substituted or unsubstitutedC₆alkyl. In certain embodiments, R^(2D) is a nitrogen protecting group,e.g., acetyl (Ac, —C═OCH₃).

In certain embodiments, R^(4D) is hydrogen. In certain embodiments,R^(4D) is substituted or unsubstituted C₁₋₆ alkyl, e.g., substituted orunsubstituted C₁alkyl, substituted or unsubstituted C₂alkyl, substitutedor unsubstituted C₃alkyl, substituted or unsubstituted C₄alkyl,substituted or unsubstituted C₅alkyl, or substituted or unsubstitutedC₆alkyl. In certain embodiments, R^(4D) is an oxygen protecting group.

In certain embodiments, R^(5D) is hydrogen. In certain embodiments,R^(5D) is substituted or unsubstituted C₁₋₆ alkyl, e.g., substituted orunsubstituted C₁alkyl, substituted or unsubstituted C₂alkyl, substitutedor unsubstituted C₃alkyl, substituted or unsubstituted C₄alkyl,substituted or unsubstituted C₅alkyl, or substituted or unsubstitutedC₆alkyl. In certain embodiments, R^(5D) is an oxygen protecting group.

In certain embodiments, both of R^(4D) and R^(5D) are hydrogen. Incertain embodiments, R^(2D) is a nitrogen protecting group, e.g., acetyl(Ac, —C═OCH₃), and R^(4D) and R^(5D) are each hydrogen.

Exemplary compounds of Formula (III) include, but are not limited to,

and salts thereof.

In Step S-2, a method of enzymatically synthesizing a compound ofFormula (III), or salt thereof, from a compound of Formula (II), or saltthereof, is performed under suitable conditions. A substrate of Formula(II) can be prepared by any method known in the art or disclosed herein.In some examples, the Formula (II) compound is isolated from thereaction mixture described in Step S-1 above. In other examples, thewhole reaction mixture of Step S-1 is used without purification of theFormula (II) compound produced therein. The Formula (II) compound can beincubated with UDP-GalNAc in the presence of a GalNAcT (e.g., abeta1,3-GalNAcT) under conditions allowing conversion of the Formula(II) compound to a Formula (III) compound. In some example, thisGalNAcT-catalyzed reaction is coupled with the UDP-GalNAc regenerationprocess as described herein. FIG. 2B. See also Examples below.

Step S-3

The third step in the biosynthetic approach (S-3) involves enzymaticconversion of a compound of Formula (III), or salt thereof, to acompound of Formula (IV), or salt thereof:

wherein R^(1A), R^(2A), R^(3A), R^(5A), R^(2B), R^(3B), R^(5B), R^(2C),R^(4C), R^(5C), R^(2D), R^(4D) and R^(5D) are as defined herein; andeach instance of R^(2E), R^(3E), R^(4E), and R^(5E) is independentlyhydrogen, substituted or unsubstituted C₁₋₆ alkyl, or an oxygenprotecting group.

In certain embodiments, R^(2E) is hydrogen. In certain embodiments,R^(2E) is substituted or unsubstituted C₁₋₆ alkyl, e.g., substituted orunsubstituted C₁alkyl, substituted or unsubstituted C₂alkyl, substitutedor unsubstituted C₃alkyl, substituted or unsubstituted C₄alkyl,substituted or unsubstituted C₅alkyl, or substituted or unsubstitutedC₆alkyl. In certain embodiments, R^(2E) is an oxygen protecting group.

In certain embodiments, R^(3E) is hydrogen. In certain embodiments,R^(3E) is substituted or unsubstituted C₁₋₆ alkyl, e.g., substituted orunsubstituted C₁alkyl, substituted or unsubstituted C₂alkyl, substitutedor unsubstituted C₃alkyl, substituted or unsubstituted C₄alkyl,substituted or unsubstituted C₅alkyl, or substituted or unsubstitutedC₆alkyl. In certain embodiments, R^(3E) is an oxygen protecting group.

In certain embodiments, R^(4E) is hydrogen. In certain embodiments,R^(4E) is substituted or unsubstituted C₁₋₆ alkyl, e.g., substituted orunsubstituted C₁alkyl, substituted or unsubstituted C₂alkyl, substitutedor unsubstituted C₃alkyl, substituted or unsubstituted C₄alkyl,substituted or unsubstituted C₅alkyl, or substituted or unsubstitutedC₆alkyl. In certain embodiments, R^(4E) is an oxygen protecting group.

In certain embodiments, R^(5E) is hydrogen. In certain embodiments,R^(5E) is substituted or unsubstituted C₁₋₆ alkyl, e.g., substituted orunsubstituted C₁alkyl, substituted or unsubstituted C₂alkyl, substitutedor unsubstituted C₃alkyl, substituted or unsubstituted C₄alkyl,substituted or unsubstituted C₅alkyl, or substituted or unsubstitutedC₆alkyl. In certain embodiments, R^(5E) is an oxygen protecting group.

In certain embodiments, R^(3E) is hydrogen. In certain embodiments,R^(3E) is substituted or unsubstituted C₁₋₆ alkyl, e.g., substituted orunsubstituted C₁alkyl, substituted or unsubstituted C₂alkyl, substitutedor unsubstituted C₃alkyl, substituted or unsubstituted C₄alkyl,substituted or unsubstituted C₅alkyl, or substituted or unsubstitutedC₆alkyl. In certain embodiments, R^(3E) is an oxygen protecting group.

In certain embodiments, each instance of R^(2E), R^(3E), R^(4E), andR^(5E) is hydrogen.

Exemplary compounds of Formula (IV) include, but are not limited to,

and salts thereof.

Step S-3 involves an enzymatic reaction via the activity of abeta1,3-galactosyltransferase, which is performed under suitableconditions known to those skilled in the art. A substrate of Formula(III), such as Gb4, can be prepared by any method known in the art ordisclosed herein. In some examples, the Formula (m) compound is isolatedfrom the reaction mixture described in Step S-2 above. In otherexamples, the whole reaction mixture of Step S-2 is used withoutpurification of the Formula (III) compound produced therein. The Formula(III) compound can be incubated with UDP-Gal in the presence of abeta1,3-galactosyltransferase under conditions allowing conversion ofthe Formula (I) compound to a Formula (IV) compound. In some example,this GalT-catalyzed reaction is coupled with the UDP-Gal regenerationprocess as described herein. FIG. 2A. See also Examples below.

In some embodiments, a beta1,3-GalNAcT/beta1,3-GalT bifunctional enzyme,such as LgtD from, e.g., H. influenza, is used in both Steps S-2 andS-3.

Step S-4

The compound of Formula (IV) may then be substituted at variouspositions on the terminal Ring E. For example, in certain embodiments ofFormula (IV), wherein R^(2E) is hydrogen, an optional step in thebiosynthetic approach (S-4) involves enzymatic conversion of a compoundof Formula (IV-a), or salt thereof, to a compound of Formula (V), orsalt thereof:

wherein R^(1A), R^(2A), R^(3A), R^(5A), R^(2B), R^(3B), R^(5B), R^(2C),R^(4C), R^(5C), R^(2D), R^(4D), R^(5D), R^(3E), R^(4E), and R^(5E) areas defined herein; and each instance of R^(1F), R^(2F), and R^(3F) isindependently hydrogen, substituted or unsubstituted C₁₋₆alkyl, or anoxygen protecting group.

In certain embodiments, R^(1F) is hydrogen. In certain embodiments,R^(1F) is substituted or unsubstituted C₁₋₆ alkyl, e.g., substituted orunsubstituted C₁alkyl, substituted or unsubstituted C₂alkyl, substitutedor unsubstituted C₃alkyl, substituted or unsubstituted C₄alkyl,substituted or unsubstituted C₅alkyl, or substituted or unsubstitutedC₆alkyl. In certain embodiments, R^(1F) is an oxygen protecting group.

In certain embodiments, R^(2F) is hydrogen. In certain embodiments,R^(2F) is substituted or unsubstituted C₁₋₆alkyl, e.g., substituted orunsubstituted C₁alkyl, substituted or unsubstituted C₂alkyl, substitutedor unsubstituted C₃alkyl, substituted or unsubstituted C₄alkyl,substituted or unsubstituted C₅alkyl, or substituted or unsubstitutedC₆alkyl. In certain embodiments, R^(2F) is an oxygen protecting group.

In certain embodiments, R^(3F) is hydrogen. In certain embodiments,R^(3F) is substituted or unsubstituted C₁₋₆alkyl, e.g., substituted orunsubstituted C₁alkyl, substituted or unsubstituted C₂alkyl, substitutedor unsubstituted C₅alkyl, substituted or unsubstituted C₄alkyl,substituted or unsubstituted C₅alkyl, or substituted or unsubstitutedC₆alkyl. In certain embodiments, R^(3F) is an oxygen protecting group.

In certain embodiments, each instance of R^(1F), R^(2F), and R^(3F) ishydrogen.

Exemplary compounds of Formula (V) include, but are not limited to,

and salts thereof.

Step S-4 involves an enzymatic reaction via the activity of analpha1,2-fucosyltransferase, which is performed under suitableconditions known to those skilled in the art. A substrate of Formula(IV), such as Gb5, can be prepared by any method known in the art ordisclosed herein. In some examples, the Formula (IV) compound isisolated from the reaction mixture described in Step S-3 above. In otherexamples, the whole reaction mixture of Step S-3 is used withoutpurification of the Formula (V) compound produced therein. The Formula(IV) compound can be incubated with GDP-Fuc in the presence of thefucosyltransferase under conditions allowing conversion of the Formula(IV) compound to a Formula (V) compound. In some example, thisFucT-catalyzed reaction is coupled with the GDP-Fuc regeneration processas described herein. FIG. 2D. See also Examples below.

Step S-5

In other embodiments of Formula (IV), wherein R^(3E) is hydrogen, anoptional step in the biosynthetic approach (S-5) involves enzymaticconversion of a compound of Formula (IV-b), or salt thereof, to acompound of Formula (VI), or salt thereof:

wherein R^(1A), R^(2A), R^(3A), R^(5A), R^(2B), R^(3B), R^(5B), R^(2C),R^(4C), R^(5C), R^(2D), R^(4D), R^(5D), R^(2E), R^(4E), and R^(5E) areas defined herein; R^(3G) is hydrogen, substituted or unsubstituted C₁₋₆alkyl, or a nitrogen protecting group; and each instance of R^(6G),R^(7G), R^(8G), and R^(9G), is independently hydrogen, substituted orunsubstituted C₁₋₆ alkyl, or an oxygen protecting group.

In certain embodiments, R^(3G) is hydrogen. In certain embodiments,R^(3G) is substituted or unsubstituted C₁₋₆alkyl, e.g., substituted orunsubstituted C₁alkyl, substituted or unsubstituted C₂alkyl, substitutedor unsubstituted C₃alkyl, substituted or unsubstituted C₄alkyl,substituted or unsubstituted C₅alkyl, or substituted or unsubstitutedC₆alkyl. In certain embodiments, R^(3G) is a nitrogen protecting group,e.g., acetyl (Ac, —C═OCH₃).

In certain embodiments, R^(6G) is hydrogen. In certain embodiments,R^(6G) is substituted or unsubstituted C₁₋₆ alkyl, e.g., substituted orunsubstituted C₁alkyl, substituted or unsubstituted C₂alkyl, substitutedor unsubstituted C₃alkyl, substituted or unsubstituted C₄alkyl,substituted or unsubstituted C₅alkyl, or substituted or unsubstitutedC₆alkyl. In certain embodiments, R^(6G) is an oxygen protecting group.

In certain embodiments, R^(7G) is hydrogen. In certain embodiments,R^(7G) is substituted or unsubstituted C₁₋₆ alkyl, e.g., substituted orunsubstituted C₁alkyl, substituted or unsubstituted C₂alkyl, substitutedor unsubstituted C₃alkyl, substituted or unsubstituted C₄alkyl,substituted or unsubstituted C₅alkyl, or substituted or unsubstitutedC₆alkyl. In certain embodiments, R^(7G) is an oxygen protecting group.

In certain embodiments, R^(8G) is hydrogen. In certain embodiments,R^(8G) is substituted or unsubstituted C₁₋₆ alkyl, e.g., substituted orunsubstituted C₁alkyl, substituted or unsubstituted C₂alkyl, substitutedor unsubstituted C₃alkyl, substituted or unsubstituted C₄alkyl,substituted or unsubstituted C₅alkyl, or substituted or unsubstitutedC₆alkyl. In certain embodiments. R^(8G) is an oxygen protecting group.

In certain embodiments, R^(9G) is hydrogen. In certain embodiments,R^(9G) is substituted or unsubstituted C₁₋₆ alkyl, e.g., substituted orunsubstituted C₁alkyl, substituted or unsubstituted C₂alkyl, substitutedor unsubstituted C₃alkyl, substituted or unsubstituted C₄alkyl,substituted or unsubstituted C₅alkyl, or substituted or unsubstitutedC₆alkyl. In certain embodiments, R^(9G) is an oxygen protecting group.

In certain embodiments, each instance of R^(6G), R^(7G), R^(8G), andR^(9G) is hydrogen. In certain embodiments, R^(3G) is a nitrogenprotecting group, e.g., acetyl (Ac, —C═OCH₃), each instance of R^(6G),R^(7G), R^(8G), and R^(9G) is hydrogen.

Exemplary compounds of Formula (V) include, but are not limited to,

and salts thereof.

Step S-5 involves an enzymatic reaction via the activity of analpha2,3-sialyltransferase, which is performed under suitable conditionsknown to those skilled in the art. A substrate of Formula (IV), such asGb5, can be prepared by any method known in the art or disclosed herein.In some examples, the Formula (IV) compound is isolated from thereaction mixture described in Step S-3 above. In other examples, thewhole reaction mixture of Step S-3 is used without purification of theFormula (IV) compound produced therein. The Formula (IV) compound can beincubated with CMP-Neu5Ac in the presence of the sialyltransferase underconditions allowing conversion of the Formula (IV) compound to a Formula(V) compound. In some example, this Sialyltransferase-catalyzed reactionis coupled with the CMP-Neu5Ac regeneration process as described herein.FIG. 2E. See also Examples below.

Each of the Steps S1-S5, as well as any combination of consecutive stepsas described above, is within the scope of this disclosure. Also withinthe scope of the present disclosure are any of the compounds produced inany of the synthesis methods described herein, e.g., those describedabove.

In some embodiments, the present disclosure features methods forsynthesizing Globo H or SSEA₄ from lactose via a chain reactioncomprising Steps S-1, S-2, S-3, and S-4 or Steps S-1, S-2, S-3, or S-5described above. The Globo H or SSEA4 can be either untailed (R^(1A)being hydrogen; see FIGS. 3 and 4), or tailed (e.g., R^(1A) being allyl;see FIGS. 5 and 6). In each step, the glycosyltransferase reaction canbe coupled with the corresponding nucleotide sugar regeneration process.FIGS. 3-6. In one example, the above-described method is performed in aone-pot manner, i.e., each prior reaction mixture is used directly forthe next step reaction without purifying the substrate produced in theprior reaction. In other words, the one-pot approach is free of any stepfor purifying any intermediate. Alternatively, Steps S-1 and S-2 areperformed in a one-spot manner without purification of any intermediate.After Step S-2, Gb4 is isolated from the reaction mixture and thepurified GB4 is used for the following Steps S3, S4, and/or S5. Nofurther purification step is performed for isolating other intermediate.

The enzymes used in each reaction step can be dissolved in each reactionmixture, or immobilized on one or more support members. When necessary,additional enzymes can be added during the chain reaction.

Enzymatic Reactors

A chain enzymatic reaction comprising any combination of two or moreconsecutive steps as described above can be performed in an enzymaticreactor, which comprises one or more reaction chambers. Each reactionchamber is designed for perform one step of the chain reaction. Inparticular, each reaction chamber comprises enzymes involved in one stepof the reaction, including each of Steps 1-S to 5-S described above.

In some embodiments, one or more enzymes, or all of the enzymes, in eachreaction chamber are immobilized on a suitable support member (e.g., asupport membrane). When necessary, reaction chambers for consecutivereaction steps can be connected such that, after termination of theenzymatic reaction in a prior reaction chamber, the resultant reactionmixture can flow into the following reaction chamber to allow the nextreaction step to occur. In some examples, the product from the priorreaction is not purified and the whole reaction mixture including theproduct is added into the next reaction chamber to allow occurrence ofthe next enzymatic reaction. See, e.g., FIGS. 3 and 4.

For example, the reaction of Step 1-S can be performed in a firstreaction chamber in the enzymatic reactor, wherein one or enzymesinvolved in Step 1-S are immobilized on a support member. Aftertermination of Step 1-S, the reaction mixture (including the Gb3product) in the first reaction chamber is placed into a second reactionchamber containing all enzymes and reagents necessary for Step 2-S forsynthesis of Gb4. In one example, the Gb4 is purified and used for thenext reaction step. In another example, the whole reaction mixture inthe second reaction chamber, including Gb4, is placed into a thirdreaction chamber that contains enzymes and reagents necessary for Step3-S, in which Gb5 is synthesized. Afterwards, the reaction mixture fromthe third reaction chamber can be placed into a fourth reaction chambercontaining enzymes and reagents necessary for Step 4-S or placed into afifth reaction chamber containing enzymes and reagents necessary forStep 5-S.

In other embodiments, the enzymatic reactor contains one reactionchamber including enzymes, reagents, and the suitable substrate,necessary for one of the synthesis steps described above. The substrateis immobilized on a support member. In one example, a reaction chambercontains the enzymes and reagents necessary for Step 1-S, in which thesubstrate, Lac-allyl, is immobilized on a support member. After Step1-S, in which Gb3-allyl is synthesized, the reaction mixture in thereaction chamber is replaced with a second reaction mixture containingenzymes and reagents necessary for Step 2-S. After synthesis ofGb4-allyl in Step 2-S, the second reaction mixture is replaced with athird reaction mixture containing enzymes and reagents for Step 3-S, inwhich Gb5-allyl is synthesized. Afterwards, the third reaction mixtureis replaced with either a fourth reaction mixture containing the enzymesand reagents for Step 4-S (for synthesis of Globo H-allyl) or a fifthreaction mixture containing the enzymes and reagents for Step 5-S (forsynthesis of SSEA₄-allyl).

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever. All publicationscited herein are incorporated by reference for the purposes or subjectmatter referenced herein.

EXAMPLES

These and other aspects of the present invention will be furtherappreciated upon consideration of the following Examples, which areintended to illustrate certain particular embodiments of the inventionbut are not intended to limit its scope, as defined by the claims.

Example 1 Synthesis of Globo-Series Oligosaccharides New Method forUDP-Gal Regeneration

In 2004, Kotake's group discovered an enzyme from Pea Sprouts, UDP-SugarPyrophosphorylase, which has broad substrate specificity towarddifferent monosaccharide-1-phosphate to form UDP-Sugar.^([19]) Two yearslater, Kotake's and Somers' groups independently published similarfunction enzyme, AtUSP, existed in Arabidopsis. ^([20],[21]) Veryrecently, the homologous enzymes also proved existing in parasites,Leishmania and Trypanosoma. ^([22],[23]) The AtUSP enzyme is interestingbecause of its intrinsic ability to condense UTP with not onlyGlc-1-phosphate and Gal-1-phosphate but also other monosaccharide-1-P,GlcA-1-phosphate, and Xyl-1-phosphate. Therefore, we chose AtUSP tocondense Gal-1-phosphate with UTP directly to render the UDP-Galregeneration and to fulfill the third regeneration of UDP-Gal synthesis.

Synthesis of Allyl-Gb3

The reaction mixture (200 mL) contained 10 mmol of allyl-lac, 10 mmol ofgalactose, 22 mmol of Phosphoenolpyruvic acid (PEP), 0.05 mmol of ATP,0.125 mmol of UTP with 10 mM MgCl₂ in 100 mM Tris-HCl buffer (pH 7.0).The reaction was initiated by addition 100 U ofα-1,4-galactosyltransferase (LgtC), 50 U of galactokinase (GalK), 150 Uof UDP-sugar pyrophosphorylase (AtUSP), 200 U of pyruvate kinase (PK)and 200 U of pyrophosphatase (PPA). The flask was incubated at 25° C.and the reaction progress was monitored by TLC, and stained byp-anisaldehyde. More enzymes were added if any of the reaction wasincomplete until the reaction was complete, and the products wereconfirmed by TLC and ESI-MS.

Synthesis of Allyl-Gb4

Following the allyl-Gb3 synthesis, additional components were added,including 9.9 mmol of N-acetylgalactosamine (GalNAc), 22 mmol of PEP,100 U of β-1,3-N-acetylgalactosaminyltransferase (β1,3GalNAcT, LgtD), 50U of N-acetylhexosamine 1-kinase (NahK), 200 U of N-acetylglucosamine1-phosphate uridylyltransferase (GlmU), 100 U of PK and 100 U of PPA, in220 mL solution. The mixture was incubated at 25° C. and monitored byTLC and ESI-MS as before until the reaction was complete. The productwas further purified by a C-18 gel column and characterized by NMR.

Synthesis of Allyl-Gb5

The reaction mixture (250 mL) contained 9 mmol of allyl-Gb4, 9 mmol ofgalactose, 22 mmol of PEP, 0.05 mmol of ATP, 0.125 mmol of UTP with 10mM MgCl₂ in 100 mM Tris-HCl buffer (pH 7.0). The reaction was initiatedby addition of 200 U of β-1,3-galactosyltransferase (β1,3GalT, LgtD), 50U of GalK, 150 U of AtUSP, 100 U of PK and 100 U of PPA and incubated at25° C., until completion.

Synthesis of Allyl-Globo H

A half amount of the reaction product of allyl-Gb5 (˜4.5 mmol) withoutadditional purification was used to produce allyl-globo H directly. Asolution containing 5 mmol of fucose, 0.05 mmol of ATP, 0.5 mmol of GTP,11 mmol PEP with 10 mM MgCl₂ in 100 mM Tris-HCl buffer (pH 7.0) wasadded 200 U of L-fucokinase/GDP-fucose pyrophosphorylase (FKP), 200 U ofPK, 200 U of PPA and 200 U of α-1,2-fucosyltransferase (FutC) incubatedat 25° C. until the reaction was complete, and the product was purifiedby C-18 gel chromatography as before and characterized.

Synthesis of Allyl-SSEA4

Another half of the allyl-Gb5 (4.5 mmol) reaction mixture was used forthe synthesis of allyl-SSEA4 by adding 5 mmol of N-acetylneuraminic acid(Neu5Ac), 0.05 mmol of ATP, 0.25 mmol of CTP, 11 mmol of PEP with 10 mMMgCl₂ in 100 mM Tris-HCl buffer (pH 8.0) followed by 50 U of Cytidinemonophosphate kinase (CMK), 120 U of CMP-sialic acid synthetase (CSS),100 U of PK, 100 U of PPA and 150 U of α-2,3-sialyltransferase(JT-FAJ-16). The progress was monitored by TLC and the product waspurified and characterized as described above.

Purification and Characterization of Oligosaccharides

Proteins in reaction mixture were removed by heating to 90° C. for 30minutes and followed by centrifugation (5000 rpm, 20 min). The filtratewas then purified by C-18 gel chromatography and eluted by a gradientfrom 100% H₂O to 10% methanol in H₂O. The fractions were collected andmonitored by TLC [butanol/ammonium hydroxide/water=5:3:2 (v/v/v)] andthe fractions with allyl-oligosaccharides were pooled and lyophilized.More than 99% purity of product could be gathered by HPLC using Cosmosil5SL-II column in (H₂O/Acetonitrile=19/81) in an isocratic mode. Thestructure of allyl-Lac, allyl-Gb3, allyl-Gb4, allyl-Gb5, allyl-Globo Hand allyl-SSEA4 were analyzed by 1H NMR, ¹³C NMR, and mass spectrometry(Avance 600 and APEX-ultra 9.4 T FTICR-MS, Bruker Daltonics).

Cloning of Genes for Nucleotide Sugar Synthesis, Glycosyltransferasesand ATP Regeneration

All genes obtained via PCR from genomic DNA or cDNA library byrespective primer (Table 5), and PCR product were ligated into themodified pET47b vector. After ATG, following are the His-tag, AcTEVprotease cutting site and ccdB positive selection gene flanked byspecial restriction recognition enzymes, or pET28a in C-terminalHis-tag. In order to increase the gene expression level, the fourglycosyltransferases were synthesized by codon optimization for E. coli.The plasmid with correct sequence was transformed into ArcticExpress/RILcompetent cell by chemical transformation method. Picked single colonyand inoculated into TB medium with kanamycin antibiotics overnight, andrefresh the cell culture into fresh TB medium, then inducing targetprotein expression by final concentration 0.1 mM IPTG when OD600 wasreaching 0.5. After that, allowed grown at 16° C. for 24 h. The E. colicells were harvested and disrupted in a buffer containing 50 mM sodiumphosphate buffer, pH8.0, 300 mM sodium chloride, and 10 mM imidazole bymicrofluidizer. Centrifuge the cell in 10,000 rpm at 4° C. for 30minutes. Then, poured the supernatant into the equilibrated Ni-NTAagarose and discard the precipitate. The bound protein was eluted in thesame buffer but containing higher concentration imidazole (250 mM). Theprotein concentration was determined by Qubit Protein Quantitation(Invitrogen, CA), and purity was confirmed by SDS-PAGE.

TABLE 5 Primers used for sialidase expressions in E. coli. Gene sourceSEQ ID Restriction from genome NO Primer^(a) Sequence (5′→3′)enzyme site or cDNA pool SEQ ID galK-F CTGTATTTTCAGGGA GCGATCGC TA AsiSIE. coli NO: 1  TGAGTCTGAAAGAAAAAACA^(b) MG1655 SEQ ID galK-RGCCTCGAGTCATTAC GTTTAAAC TC PmeI ATCC NO: 2  AGCACTGTCCTGCTCCTTG 700926SEQ ID atusp-F CTGTATTTTCAGGGA GCGATCGC TA AsiSI cDNA NO: 3 TGGCTTCTACGGTTGATTC pool of SEQ ID atusp-R GCCTCGAGTCATTAC GTTTAAAC TCPmeI Arabidopsis NO: 4  AATCTTCAACAGAAAATTTGC thaliana SEQ ID lgtC-F^(b)GATATA CCATGG AAATGGACATCGT NcoI Gene NO: 5  TTTCGCGGCG optimizationSEQ ID lgtC-R^(b) GTGGTG CTCGAG GTAGATTTTACGC XhoI NO: 6  AGGAAACGSEQ ID nahK-F CTGTATTTTCAGGGAGCGATCGCTA AsiSI Bifidobacterium NO: 7 TGAACAAGACTTATGATTTTAAAAG longum SEQ ID nahK-R GCCTCGAGTCATTACGTTTAAACTTPmeI Atcc NO: 8  AAATGTATGAATATACTATCTTC 15697 SEQ ID glmU-FCTGTATTTTCAGGGA GCGATCGC TA AsiSI E. coli NO: 9  TGTTGAATAATGCTATGAGCMG1655 SEQ ID glmU-R GCCTCGAGTCATTAC GTTTAAAC TC PmeI ATCC NO: 10ACTTTTTCTTTACCGGACG 700926 SEQ ID lgtD-F^(b) GATATA CCATGG AAAACTGCCCGCTNcoI Gene NO: 11 GGTTTCT optimization SEQ ID lgtD-R^(b) GTGGTG CTCGAGGAAGATAACGTTG XhoI NO: 12 ATTTTACGG SEQ ID fkp-F CAGGGA GCGATCGCTATGCAAAAAC AsiSI Bacteroides NO: 13 TACTATCTTTA fragilis 9343 SEQ IDfkp-R CATTAC GTTTAAAC TTATGATCGTG PmeI ATCC NO: 14 ATACTTGGAA 25285SEQ ID futC-F^(b) CTGTATTTTCAGGGA GCGATCGC TA AsiSI Gene NO: 15TGGCGTTCAAAGTTGTTCAG optimization SEQ ID futC-R^(b) GCCTCGAGTCATTACGTTTAAAC TT PmeI NO: 16 ACGCGTTGTATTTCTGAGAT SEQ ID cmk-F CAGGGAGCGATCGC TATGACGGCAA AsiSI E. coli NO: 17 TTGCCCCGGTT MG1655 SEQ IDcmk-R CATTAC GTTTAAAC TTATGCGAGAG PmeI ATCC NO: 18 CCAATTTCTG 700926SEQ ID CSS-F GATATA CCATGG AAACAAATATTGC NcoI Pasteurella NO: 19GATCATTCCTG multocida SEQ ID CSS-R GTGGTG CTCGAG TTTATTGGATAAA XhoIATCC BAA- NO: 20 ATTTCCGCGAG 1113 SEQ ID jt-faj- GATATA CCATGGAAATGAACAACGA NcoI Gene NO: 21 16-F^(b) CAACTCTACC optimization SEQ IDjt-faj- GTGGTG CTCGAG GATGTCAGAGATC XhoI NO: 22 16-R^(b) AGTTTGATGSEQ ID pykF-F CTGTATTTTCAGGGA GCGATCGC TA AsiSI E. coli NO: 23TGAAAAAGACCAAAATTGTTTG MG1655 SEQ ID pykF-R GCCTCGAGTCATTAC GTTTAAAC TTPmeI ATCC NO: 24 ACAGGACGTGAACAGATG 700926 SEQ ID ppa-F CAGGGA GCGATCGCTATGAGCTTAC AsiSI E. coli NO: 25 TCAACGTCCCT MG1655 SEQ ID ppa-R CATTACGTTTAAAC TTATTTATTCT PmeI ATCC NO: 26 TTGCGCGCTC 700926 ^(a)apair ofprimers for forward (F) and reversed (R) PCR reactions to amplify thecoding sequence of each gene. ^(b)Underline with bold means the site ofrestriction enzyme recognition. ^(c)Codon optimization for E. coli. See,e.g., Puigbò et al., Nucleic Acids Research (2007) 35(S2):W126-W130

Enzyme Assay

In order to maintain constant assay conditions, all activity wasmeasured at 37° C. with 10 mM MgCl₂, 100 mM Tris, and at a pH of 7.5.

(i) Measurement of the Galactokinase (GalK), N-Acetylhexosamine Kinase(NahK), Fucokinase (FKP) and Cytidine Monophosphate Kinase (CMK)Activity

The fluorometric assay method was based on monitor of ADP production(ATP consumption) by using the pyruvate kinase/lactate dehydrogenasecoupled enzymatic assay for the NADH consumption. See, e.g., Murray etal., “Mechanism of Human α-1,3-Fucosyltransferase V: Glycosidic CleavageOccurs Prior to Nucleophilic Attack” Biochemistry (1997) 36:823-831; andGosselin et al., “A Continuous Spectrophotometric Assay forGlycosyltransferases” Analytical Biochemistry (1994) 220:92-97.Fluorescence property of NADH has an excitation wavelength of 340 nm andan emission wavelength of 450 nm. A 100 uL of reaction mixture wasprepared containing the coupling enzyme (5 units of pyruvate kinase and7 units of lactic dehydrogenase from rabbit muscle) and substrates andcofactors (0.2 mM NADH, 0.8 mM PEP, 10 mM MgCl₂) in 100 mM Tris (pH7.5). Reactions were initiated by the addition of the respective sugar.The kinetic parameters, K_(cat) and K_(m) were calculated by curvefitting the experimental data with the theoretical equation, usingGrafit version 7 (Erithacus Software, Middlesex, UK). One unit of sugarkinase activity is defined as 1 umol of sugar-1-P formation per minuteat 25° C.

(ii) Measurement of UDP-Sugar Pyrophosphorylase (AtUSP), N-AcetylGlucosamine-1-Phosphate Uridyltransferase (GlmU), GDP-L-FucosePyrophosphorylase (FKP) and CMP-Sialic Acid Synthetases (CSS) Activity

The production of pyrophosphate was measured using the EnzCheckPyrophosphate Assay Kit (Invitrogen, CA, USA). Assay componentsincluding: 200 uM 2-amino-6-mercapto-7-methylpurine ribonucleoside, 1unit nucleoside phosphorylase, 0.03 unit inorganic pyrophosphatase, 10mM MgCl₂, 50 mM Tris, pH 7.5 in 100 uL scale in UV-Star microplates(Greiner Bio One, Germany). All components except FKP were mixed in themicroplates and allowed to equilibrate until a flat baseline wasachieved. Reactions were initiated by the addition of enzyme. One unitof enzyme activity is defined as the producing 1 umol of nucleotidesugar from the respective sugar-1-Ps per minute at 25° C., except forCMP-sialic acid synthetase, which is defined as 1 umol of pyrophosphateformation per minute at 25° C.

(iii) Measurement of glycosyltransferase: α-1,4-galactosyltransferase(LgtC), β1,3-N-acetylgalactosaminyltransferase (β1,3GalNAcT, LgtD),β-1,3-galactosyltransferase (LgtD), α-1,2-fucosyltransferase (FutC) andα-2,3-sialyltransferase (JT-FAJ-16).

The fluorometric assay method monitored UDP, GDP, or CDP productionusing the pyruvate kinase/lactate dehydrogenase coupled enzymatic assayfor the NADH consumption. See, e.g., Murray et al., “Mechanism of Humanα-1,3-Fucosyltransferase V: Glycosidic Cleavage Occurs Prior toNucleophilic Attack” Biochemistry (1997) 36:823-831; and Gosselin etal., “A Continuous Spectrophotometric Assay for Glycosyltransferases”Analytical Biochemistry (1994) 220:92-97. The assay components exceptnucleotide sugar were simultaneously incubated in the multiple platefluorometer (SpectraMax M2 Readers, Molecular Devices) at 25° C.Reactions were initiated by the addition of corresponding nucleotidesugar. The kinetic parameters, K_(cat) and K_(m) were calculated bycurve fitting the experimental data with the theoretical equation, usingGrafit version 7 (Erithacus Software, Middlesex, UK). One unit ofactivity is defined as the amount of enzyme that catalyzes the transfer1 umol sugar from respective nucleotide sugar to acceptor per minute at25° C.

(iv) Measurement of Pyruvate Kinase (PyrK)

Pyruvate kinase assay was slightly modified from sugar kinasemeasurement previous mentioned, also based on NADH consumption. A 100 uLof reaction mixture is prepared containing 0.8 mM ADP, 0.8 mM PEP, 0.2mM NADH, 10 mM MgCl₂, and 5 units of lactic dehydrogenase from rabbitmuscle in 100 mM Tris (pH 7.5) in black multiplate. NADH has anexcitation wavelength at 340 nm and an emission wavelength at 450 nm.Reaction is initiated by adding a suitable amount of recombinant E. colipyruvate kinase. One unit of pyruvate kinase is defined as conversion of1.0 μmole of phospho(enol)pyruvate to pyruvate per minute at 25° C.

(v) Measurement of Pyrophosphatase (PPA)

Pyrophosphatase assay is slightly modified from pyrophorylase protocolfrom commercial kit EnzCheck Pyrophosphate Assay Kit (Invitrogen, CA,USA). Assay components including: 1 mM pyrophosphate, 200 uM2-amino-6-mercapto-7-methylpurine ribonucleoside, 1 unit nucleosidephosphorylase, 10 mM MgCl₂, 50 mM Tris, at a pH of 7.5 in 100 uL scalein UV-Star microplates (Greiner Bio One, Germany) with suitable amountof recombinant E. coli pyrophosphatase. One unit of pyrophosphataseactivity is defined as liberation of 1.0 umole of inorganicpyrophosphate per minute at 25° C.

(vi) Measurement of Optimum pH

The optimum pH for enzyme activity was determined in the standard enzymeassay mentioned above in the pH range 4.0-10.0, including sodiumacetate, MES, MOPS, HEPES, Tris-HCl, CHES buffer. The pH of the bufferwas adjusted at the temperature of incubation. All reactions wereperformed in triplicate for statistical evaluation.

(vii) Measurement of Optimum Divalent Metal Ion

The assay for metal requirement was conducted in standard assaycondition. Enzymes were mixed with metal ion (Mg²⁺, Mn²⁺, Mg²⁺, Mn²⁺,Ca²⁺, Zn²⁺, Co²⁺, or Ni²⁺) in a final concentration of 10 mM, in thepresence and absence of EDTA. All reactions were performed in triplicatefor statistical evaluation.

(viii) Measurement of Optimum Temperature

The effect of temperature on the activity of enzymes were determined byincubating an appropriate amount of purified enzyme in MOPS buffer (pH7.0), 10 mM MgCl₂ and respective substrates. In order to keep the assayconsist, all components were mixed well and preheated at assaytemperature for 5 min, and the reaction was started by adding the enzymeand recorded by multimode plate readers (SpectraMax MS, MolecularDevices) in constant temperature. The temperature ranged from 20 to 60°C. All reactions were performed in triplicate for statisticalevaluation.

Enzyme Composition UDP-Gal Regeneration/Galactosylation

-   -   1. GalK: galactokinase, from E. coli    -   2. AtUSP: UDP-sugar pyrophosphorylase from Arabidopsis thaliana    -   3. LgtC: α1,4galactosyltransferase, from Neisseria meningitidis,        but codon optimization for E. coli    -   4. PykF: pyruvate kinase, from E. coli    -   5. PPA: pyrophosphatase, from E. coli

The coding sequence of the coden-optimized LgtC enzyme is provided below(SEQ ID NO: 27):

ATGGACATCGTTTTCGCGGCGGACGACAACTACGCGGCGTACCTGTGCGTTGCGGCGAAATCTGTTGAAGCGGCGCACCCGGACACCGAAATCCGTTTCCACGTTCTGGACGCGGGTATCTCTGAAGCGAACCGTGCGGCGGTTGCGGCGAACCTGCGTGGTGGTGGTGGTAACATCCGTTTCATCGACGTTAACCCGGAAGACTTCGCGGGTTTCCCGCTGAACATCCGTCACATCTCTATCACCACCTACGCGCGTCTGAAACTGGGTGAATACATCGCGGACTGCGACAAAGTTCTGTACCTGGACATCGACGTTCTGGTTCGTGACTCTCTGACCCCGCTGTGGGACACCGACCTGGGTGACAACTGGCTGGGTGCGTGCATCGACCTGTTCGTTGAACGTCAGGAAGGTTACAAACAGAAAATCGGTATGGCGGACGGTGAATACTACTTCAACGCGGGTGTTCTGCTGATCAACCTGAAAAAATGGCGTCGTCACGACATCTTCAAAATGTCTTGCGAATGGGTTGAACAGTACAAAGACGTTATGCAGTACCAGGACCAGGACATCCTGAACGGTCTGTTCAAAGGTGGTGTTTGCTACGCGAACTCTCGTTTCAACTTCATGCCGACCAACTACGCGTTCATGGCGAACCGTTTCGCGTCTCGTCACACCGACCCGCTGTACCGTGACCGTACCAACACCGTTATGCCGGTTGCGGTTTCTCACTACTGCGGTCCGGCGAAACCGTGGCACCGTGACTGCACCGCGTGGGGTGCGGAACGTTTCACCGAACTGGCGGGTTCTCTGACCACCGTTCCGGAAGAATGGCGTGGTAAACTGGCGGTTCCGCACCGTATGTTCTCTACCAAACGTATGCTGCAGCGTTGGCGTCGTAAACTGTCTGCGCGTTTCCTGCGTAAAATCTACTGA

UDP-GalNAc Regeneration/Acetylgalactosamination

-   -   1. GalNAcK: N-Acetylliexosarnine 1-Kinases, from B. longum    -   2. GlmU: N-acetylglucosamine 1-phosphate uridylyltransferase        from E. coli    -   3. LgtD: β1,3galactosyltransferase, from Haemophilus influenza,        but codon optimization for E. coli    -   4. PykF: pyruvate kinase, from E. coli    -   5. PPA: pyrophosphatase, from E. coli

The coding sequence of the coden-optimized LgtD enzyme is provided below(SEQ ID) NO: 28):

ATGGAAAACTGCCCGCTGGTTTCTGTTATCGTTTGCGCGTACAACGCGGAACAGTACATCGACGAATCTATCTCTTCTATCATCAACCAGACCTACGAAAACCTGGAAATCATCGTTATCAACGACGGTTCTACCGACCTGACCCTGTCTCACCTGGAAGAAATCTCTAAACTGGACAAACGTATCAAAATCATCTCTAACAAATACAACCTGGGTTTCATCAACTCTCTGAACATCGGTCTGGGTTGCTTCTCTGGTAAATACTTCGCGCGTATGGACGCGGACGACATCGCGAAACCGTCTTGGATCGAAAAAATCGTTACCTACCTGGAAAAAAACGACCACATCACCGCGATGGGTTCTTACCTGGAAATCATCGTTGAAAAAGAATGCGGTATCATCGGTTCTCAGTACAAAACCGGTGACATCTGGAAAAACCCGCTGCTGCACAACGACATCTGCGAAGCGATGCTGTTCTACAACCCGATCCACAACAACACCATGATCATGCGTGCGAACGTTTACCGTGAACACAAACTGATCTTCAACAAAGACTACCCGTACGCGGAAGACTACAAATTCTGGTCTGAAGTTTCTCGTCTGGGTTGCCTGGCGAACTACCCGGAAGCGCTGGTTAAATACCGTCTGCACGGTAACCAGACCTCTTCTGTTTACAACCACGAACAGAACGAAACCGCGAAAAAAATCAAACGTGAAAACATCACCTACTACCTGAACAAAATCGGTATCGACATCAAAGTTATCAACTCTGTTTCTCTGCTGGAAATCTACCACGTTGACAAATCTAACAAAGTTCTGAAATCTATCCTGTACGAAATGTACATGTCTCTGGACAAATACACCATCACCTCTCTGCTGCACTTCATCAAATACCACCTGGAACTGTTCGACCTGAAACAGAACCTGAAAATCATCAAAAAATTCATCCGTAAAATCAAC GTTATCTTCTAG

GDP-FK Regeneration/Fucosylation

-   -   1. FKP: L-fucokinase/GDP-fucose pyrophosphorylase, from        Bacteroides fragilis    -   2. FutC: α1,2fucosyltransferase, from Helicobacter, pylor, but        codon optimization for E. coli    -   3. PykF: pyruvate kinase, from E. coli    -   4. PPA: pyrophosphatase, from E. coli

The coding sequence of the coden-optimized FutC enzyme is provided below(SEQ ID NO: 29):

ATGGCGTTCAAAGTTGTTCAGATCTGCGGTGGTCTGGGTAACCAGATGTTCCAGTACGCGTTCGCGAAATCTCTGCAGAAACACTCTAACACCCCGGTTCTGCTGGACATCACCTCTTTCGACTGGTCTGACCGTAAAATGCAGCTGGAACTGTTCCCGATCGACCTGCCGTACGCGTCTGCGAAAGAAATCGCGATCGCGAAAATGCAGCACCTGCCGAAACTGGTTCGTGACGCGCTGAAATGCATGGGTTTCGACCGTGTTTCTCAGGAAATCGTTTTCGAATACGAACCGAAACTGCTGAAACCGTCTCGTCTGACCTACTTCTTCGGTTACTTCCAGGACCCGCGTTACTTCGACGCGATCTCTCCGCTGATCAAACAGACCTTCACCCTGCCGCCGCCGCCGGAAAACAACAAAAACAACAACAAAAAAGAAGAAGAATACCAGTGCAAACTGTCTCTGATCCTGGCGGCGAAAAACTCTGTTTTCGTTCACATCCGTCGTGGTGACTACGTTGGTATCGGTTGCCAGCTGGGTATCGACTACCAGAAAAAAGCGCTGGAATACATGGCGAAACGTGTTCCGAACATGGAACTGTTCGTTTTCTGCGAAGACCTGGAATTCACCCAGAACCTGGACCTGGGTTACCCGTTCATGGACATGACCACCCGTGACAAAGAAGAAGAAGCGTACTGGGACATGCTGCTGATGCAGTCTTGCCAGCACGGTATCATCGCGAACTCTACCTACTCTTGGTGGGCGGCGTACCTGATCGAAAACCCGGAAAAAATCATCATCGGTCCGAAACACTGGCTGTTCGGTCACGAAAACATCCTGTGCAAAGAATGGGTTAAAATCGAATCTCACTTCGAAGTTAAATCTCAGAAATACAACGCGTAA

CMP-Neu5Ac Regeneration/Sialylation

-   -   1. CMK: Cytidine monophosphate kinase, from E. coli    -   2. Css: CMP-sialic acid synthetase, from Pasteurella multocida    -   3. JT-FAJ-16: α2,3sialyltransferase, from marine bacteria, but        codon optimization for E. coli    -   4. PykF: pyruvate kinase, from E. coli    -   5. PPA: pyrophosphatase, from E. coli

The coding sequence of the coden-optimized JT-FAJ-16 enzyme is providedbelow (SEQ ID NO: 30):

Materials and Chemicals

ATGAACAACGACAACTCTACCACCACCAACAACAACGCGATCGAAATCTACGTTGACCGTGCGACCCTGCCGACCATCCAGCAGATGACCAAAATCGTTTCTCAGAAAACCTCTAACAAAAAACTGATCTCTTGGTCTCGTTACCCGATCACCGACAAATCTCTGCTGAAAAAAATCAACGCGGAATTCTTCAAAGAACAGTTCGAACTGACCGAATCTCTGAAAAACATCATCCTGTCTGAAAACATCGACAACCTGATCATCCACGGTAACACCCTGTGGTCTATCGACGTTGTTGACATCATCAAAGAAGTTAACCTGCTGGGTAAAAACATCCCGATCGAACTGCACTTCTACGACGACGGTTCTGCGGAATACGTTCGTATCTACGAATTCTCTAAACTGCCGGAATCTGAACAGAAATACAAAACCTCTCTGTCTAAAAACAACATCAAATTCTCTATCGACGGTACCGACTCTTTCAAAAACACCATCGAAAACATCTACGGTTTCTCTCAGCTGTACCCGACCACCTACCACATGCTGCGTGCGGACATCTTCGACACCACCCTGAAAATCAACCCGCTGCGTGAACTGCTGTCTAACAACATCAAACAGATGAAATGGGACTACTTCAAAGACTTCAACTACAAACAGAAAGACATCTTCTACTCTCTGACCAACTTCAACCCGAAAGAAATCCAGGAAGACTTCAACAAAAACTCTAACAAAAACTTCATCTTCATCGGTTCTAACTCTGCGACCGCGACCGCGGAAGAACAGATCAACATCATCTCTGAAGCGAAAAAAGAAAACTCTTCTATCATCACCAACTCTATCTCTGACTACGACCTGTTCTTCAAAGGTCACCCGTCTGCGACCTTCAACGAACAGATCATCAACGCGCACGACATGATCGAAATCAACAACAAAATCCCGTTCGAAGCGCTGATCATGACCGGTATCCTGCCGGACGCGGTTGGTGGTATGGGTTCTTCTGTTTTCTTCTCTATCCCGAAAGAAGTTAAAAACAAATTCGTTTTCTACAAATCTGGTACCGACATCGAAAACAACTCTCTGATCCAGGTTATGCTGAAACTGAACCTGATCAACCGTGACAACATCAAACTGATCTCTGACATCTAA

All nucleotide, sugar, nucleotide sugar and chemicals were purchasedfrom Sigma-Aldrich (St. Louis, Mo.). Restriction enzyme and T4 DNAligase acquired from NEB (Beverly, Mass.). Primer ordered from ProligoSingapore Pte Ltd (Singapore). Ni-NTA Agarose obtained from Qiagen(Santa Clarita, Calif.). Bio-Gel P2 gel was purchase from Bio-Rad(Hercules, Calif.). Plasmid pET28a, pET47b and precoated glass platesTLC covered in Silica Gel 60, F254 with 0.25 mm layer thickness waspurchase from EMD Chemicals Inc (Carlsbad, Calif.) were purchased fromEMD Chemicals Inc (Carlsbad, Calif.). ArcticExpress/RIL competent cellwere obtained from Agilent Genomics (La Jolla, Calif.). All othermaterials not mentioned above were purchased as high quality aspossible.

All reactions were monitored by thin-layer chromatography. (mobilephase: Butanol:acetate:water=5:3:2). Staining the TLC by p-Anisaldehyde.

Synthesis of Allyl-Lac

The synthesis of different lactose with linker was followed by theliterature reported method [Carbohydrate Research 2004, 339,2415-2424.]. ¹H NMR (600 MHz, D2O) δ 6.01 (m, 1H), 5.40-5.37 (dd,J=17.3, 1.4 Hz, 1H), 5.30-5.28 (d, J=10.3 Hz, 1H), 4.54 (d, J=8.1 Hz,1H), 4.46 (d, J=7.8 Hz, 1H), 4.41-4.38 (m, 1H), 4.25-4.22 (m, 1H),4.00-3.97 (dd, J=12.3, 2.1 Hz, 1H), 3.93 (d, J=3.3 Hz, 1H), 3.80-3.71(m, 4H), 3.67-3.53 (m, 5H), 3.35-3.33 (m, 1H); ¹³C NMR (150 MHz, D2O) δ133.3, 118.7, 102.9, 101.0, 78.3, 75.3, 74.7, 74.4, 72.8, 72.5, 70.9,70.6, 68.5, 60.9, 60.1; HRMS (ESI-TOF, MNa⁺) C₁₅H₂₆O₁₁Na⁺ calcd for405.1367. found 405.1346.

Large Scale Production of Gb3 with Linker

5 mmol lactose with linker, 5 mmol galactose, 12 mmol Phosphoenolpyruvicacid (PEP), 0.25 mmol ATP, 0.25 mmol UTP and 10 mM MgCl₂ were added into100 mM Tris-HCl buffer (pH 7.5) solution. The reaction was initiated byaddition suitable amount of α-1,4-galactosyltransferase (LgtC),galactokinase (GalK), UDP-sugar pyrophosphorylase (AtUSP), pyruvatekinase (PK) and pyrophosphatase (PPA). The flask was placed into anincubator at 16-50° C. with gentle shaking. The reaction was monitoredby TLC. More enzymes are added if the reaction stops. The reaction isstopped when no more starting material is observed by TLC. The Gb3product was isolated by ¹⁸C reverse phase column in 99% yield.

Allyl-Gb3: ¹H NMR (600 MHz, D₂O) δ 6.00 (m, 1H), 5.42 (d, J=17.2 Hz,1H), 5.32 (d, J=10.4 Hz, 1H), 4.97 (d, J=3.3 Hz, 1H), 4.56 (d, J=7.9 Hz,1H), 4.53 (d, J=7.7 Hz, 1H), 4.43-4.37 (m, 2H), 4.27-4.24 (m, 1H),4.06-3.58 (m, 16H), 3.37-3.34 (t, J=8.0 Hz, 1H); ¹³C NMR (150 MHz, D2O)δ 133.3, 118.7, 103.3, 100.9, 100.3, 78.6, 77.3, 75.4, 74.8, 74.5, 72.9,72.2, 70.9, 70.8, 70.6, 69.1, 68.9, 68.5, 60.5, 60.4, 60.0; HRMS(ESI-TOF, MNa⁺) C₂₁H₃₆O₁₆Na⁺ calcd for 567.1896. found 567.1858.

¹H NMR (600 MHz, D₂O) δ 4.97 (d, J=3.3 Hz, 1H), 4.56 (d, J=7.9 Hz, 1H),4.54-4.50 (m, 2H), 4.37 (dd, J=6.0 Hz, J=0.6 Hz, 1H), 4.27-4.24 (m, 1H),4.06-3.59 (m, 18H), 3.35 (t, J=8.0 Hz, 1H), 3.03 (t, J=7.4 Hz, 2H),1.75-1.68 (m, 4H), 1.51-1.46 (m, 2H); ¹³C NMR (150 MHz, D2O) δ 103.3,101.9, 100.3, 78.7, 77.3, 75.4, 74.8, 74.5, 72.9, 72.2, 70.9, 70.8,70.6, 69.1, 68.9, 68.5, 60.5, 60.4, 60.0, 39.3, 28.1, 26.4, 22.1.

Large Scale Production of Gb4 with Linker

5 mmol Gb3 with linker, 5 mmol N-acetylgalactosamine (GalNAc), 12 mmolPhosphoenolpyruvic acid (PEP), 0.25 mmol ATP, 0.25 mmol UTP and 10 mMMgCl₂ were added into 100 mM Tris-HCl buffer (pH 7.5) solution. Thereaction was initiated by addition suitable amount ofβ-1,3-N-acetylgalactosaminyltransferase (LgtD), N-acetylhexosamine1-kinase (NahK), N-acetylglucosamine 1-phosphate uridylyltransferase(GlmU), pyruvate kinase (PK) and pyrophosphatase (PPA). The flask wasplaced into an incubator at 16-50° C. with gentle shaking. The reactionwas monitored by TLC. More enzymes are added if the reaction stops. Thereaction is stopped when no more starting material is observed by TLC.The Gb4 product was isolated by ¹⁸C reverse phase column in 96% yield.

Allyl-Gb4: ¹H NMR (600 MHz, D₂O) δ 6.01 (m, 1H), 5.40-5.38 (dd, J=17.3,1.4 Hz, 1H), 5.30 (d, J=10.5 Hz, 1H), 4.92 (d, J=3.9 Hz, 1H), 4.64 (d,J=8.5 Hz, 1H), 4.54 (d, J=7.9 Hz, 1H), 4.53 (d, J=7.8 Hz, 1H), 4.42-4.38(m, 2H), 4.26-4.22 (m, 2H), 4.05 (d, J=2.9 Hz, 1H), 4.01-3.99 (dd,J=12.3, 1.8 Hz, 1H), 3.98-3.89 (m, 5H), 3.86-3.74 (m, 7H), 3.72-3.57 (m,7H), 3.37-3.34 (t, J=8.6 Hz, 1H), 2.05 (s, 3H); ¹³C NMR (150 MHz, D2O) δ133.2, 118.7, 103.3, 103.2, 100.9, 100.4, 78.7, 78.6, 77.2, 75.4, 74.9,74.8, 74.5, 72.9, 72.1, 70.9, 70.8, 70.6, 70.2, 68.9, 67.7, 67.6, 60.9,60.5, 60.3, 60.2, 60.0, 52.6, 22.2; HRMS (MALDI, MNa⁺) C₂₉H₄₉NO₂₁Na⁺calcd for 770.2689. found 770.2654.

Large Scale Production of Gb5 with Linker

5 mmol allyl-Gb4, 5 mmol galactose, 12 mmol Phosphoenolpyruvic acid(PEP), 0.25 mmol ATP, 0.25 mmol UTP with 10 mM MgCl₂ were added into 100mM Tris-HCl buffer (pH 7.5). The reaction was initiated by additionsuitable amount of β-1,3-galactosyltransferase, galactokinase (GalK),UDP-sugar pyrophosphorylase (AtUSP), pyruvate kinase (PK) andpyrophosphatase (PPA). The flask was placed into an incubator at 16-50°C. with gentle shaking. The reaction was monitored by TLC. More enzymesare added if the reaction stops. The reaction is stopped when no morestarting material is observed by TLC. The Gb5 product was purified by¹⁸C reverse phase column in 95% yield.

Allyl-Gb5: ¹H NMR (600 MHz, D₂O) δ 6.01 (m, 1H), 5.41-5.38 (dd, J=17.3,1.4 Hz, 1H), 5.31 (d, J=10.6 Hz, 1H), 4.93 (d, J=4.0 Hz, 1H), 4.71 (d,J=8.5 Hz, 1H), 4.55 (d, J=8.1 Hz, 1H), 4.53 (d, J=7.8 Hz, 1H), 4.47 (d,J=7.7 Hz, 1H), 4.42-4.39 (m, 2H), 4.27-4.23 (m, 2H), 4.20 (d, J=3.2 Hz,1H), 4.09-3.90 (m, 8H), 3.87-3.59 (m, 17H), 3.55-3.52 (m, 1H), 3.36-3.33(t, J=8.6 Hz, 1H), 2.04 (s, 3H); ¹³C NMR (150 MHz, D2O) δ 175.1, 133.2,118.7, 104.8, 103.3, 102.9, 100.9, 100.4, 79.6, 78.7, 78.6, 77.2, 75.4,74.9, 74.8, 74.6, 74.5, 72.9, 72.4, 72.1, 70.9, 70.6 (2C), 70.2, 68.9,68.5, 67.9, 67.6, 60.9 (2C), 60.33, 60.28, 60.0, 51.5, 22.2; HRMS(ESI-TOF, MNa⁺) C₃₅H₅₉NO₂₆Na⁺ calcd for 932.3218. found 932.3235.

¹H NMR (600 MHz, D₂O), 4.47 (d, 1H, J=8.42 Hz), 4.30 (d, 1H, J=7.9 Hz),4.28 (d, 1H, J=8.1 Hz), 4.24 (d, 1H, J=7.7 Hz), 4.19 (t, 1H J=7.0 Hz),4.04 (d, 1H, J=2.8 Hz), 3.97 (d, 1H, J=2.98 Hz), 3.87-3.35 (m, 32H),3.30 (t, 1H, J=7.7 Hz), 3.09 (t, 1H, J=8.5 Hz), 2.79 (t, 2H, J=7.6 Hz),1.82 (s, 3H), 1.51-1.43, (m, 4H), 1.28-1.21 (m, 2H) ¹³C NMR (150 MHz,D₂O), δ 175.0, 104.7, 103.1, 102.8, 101.8, 100.2, 79.4, 78.5, 78.4,76.9, 75.3, 74.8, 74.7, 74.4, 74.3, 72.8, 72.2, 71.9, 70.6, 70.4, 70.0,69.9, 68.7, 68.4, 67.8, 67.4, 60.82, 60.77, 60.13, 60.1, 59.8, 51.3,39.1, 28.0, 26.3, 22.1, 21.9 MALDI-TOF: C₃₇H₆₆N₂O₂₆ [M+H]⁺ calculated955.3904. found 955.3972.

Large Scale Production of Globo H with Linker

5 mmol Gb5 with linker, 5 mmol fucose, 12 mmol Phosphoenolpyruvic acid(PEP), 0.25 mmol ATP, 0.25 mmol GTP with 10 mM MgCl₂ were added into 100mM Tris-HCl buffer (pH 7.5). The reaction was initiated by additionsuitable amount of α-1,2-fucosyltransferase, L-fucokinase/GDP-fucosepyrophosphorylase (FKP), pyruvate kinase (PK) and pyrophosphatase (PPA).The flask was placed into an incubator at 16-50° C. with gentle shaking.The reaction was monitored by TLC. More enzymes are added if thereaction stops. The reaction is stopped when no more starting materialis observed by TLC. The Globo H product was purified by ¹⁸C reversephase column in 94% yield.

Allyl-Globo H: ¹H NMR (600 MHz, D₂O) δ 6.01 (m, 1H), 5.41-5.38 (dd,J=17.3, 1.4 Hz, 1H), 5.31 (d, J=10.7 Hz, 1H), 5.24 (d, J=4.0 Hz, 1H),4.91 (d, J=3.9 Hz, 1H), 4.63 (d, J=7.7 Hz, 1H), 4.56-4.52 (m, 3H),4.42-4.40 (m, 2H), 4.26-4.23 (m, 3H), 4.12 (d, J=2.2 Hz, 1H), 4.05 (d,J=3.0 Hz, 1H), 4.03-3.59 (m, 28H), 3.36-3.33 (t, J=8.2 Hz, 1H), 2.06 (s,3H), 1.24 (d, J=6.5 Hz, 3H); ¹³C NMR (150 MHz, D2O) δ 174.3, 133.2,118.7, 103.9, 103.2, 102.0, 100.9, 100.4, 99.3, 78.7, 78.3, 77.1, 76.3,76.1, 75.5, 75.0, 74.8, 74.6, 74.5, 73.5, 72.9, 72.1, 71.8, 70.8, 70.6,70.1, 69.5, 69.2, 69.1, 68.5, 68.0, 67.8, 66.8, 60.95, 60.93, 60.3 (2C),60.0, 51.6, 22.2, 15.3; HRMS (MALDI, MNa+) C₄₁H₇₀NO₃₀Na⁺ calcd for1079.3875. found 1078.4145.

¹H NMR (600 MHz, D₂O) δ 5.12 (d, 1H, J=3.9 Hz), 4.78 (d, 1H, J=3.6 Hz),4.50 (d, 1H, J=7.7 Hz), 4.43 (d, 1H, J=7.5 Hz), 4.40 (d, 1H, J=7.7 Hz),4.37 (d, 1H, J=8.0 Hz), 4.30 (t, 1H, J=6.2 Hz), 4.15-4.10 (m, 2H), 3.99(d, 1H, J=1.8 Hz), 3.92 (d, 1H, J=2.2 Hz), 3.90-3.47 (m, 33H), 3.19 (t,1H, J=8.3 Hz), 2.89 (t, 2H, J=7.5 Hz), 1.94 (s, 3H), 1.60-1.55 (m, 4H),1.38-1.31 (m, 2H), 1.11 (d, 3H, J=6.4 Hz). ¹³C NMR (150 MHz, D₂O) δ176.1, 105.7, 105.0, 103.74, 103.65, 102.1, 100.97, 80.5, 79.9, 78.8,78.0, 77.8, 77.2, 76.76, 76.5, 76.3, 76.2, 75.3, 74.6, 73.8, 73.5, 72.5,71.81, 71.78, 71.2, 71.1, 70.9, 70.8, 70.2, 69.7, 69.5, 68.5, 62.66,62.64, 62.0, 61.7, 53.3, 41.0, 29.9, 28.1, 23.9, 23.8, 17.0 MALDI-TOF:C₄₃H₇₆N₂O₃₀ [M+Na]⁺ calculated 1123.4381. found 1123.4385.

Large Scale Production of SSEA4 with Linker

5 mmol Gb5 with linker, 5 mmol fucose, 12 mmol phosphoenolpyruvic acid(PEP), 0.25 mmol ATP, 0.25 mmol CTP with 10 mM MgCl₂ were added into 100mM Tris-HCl buffer (pH 7.5). The reaction was initiated by additionsuitable amount of α-2,3-sialyltransferase, cytidine monophosphatekinase (CMK), CMP-sialic acid synthetase (CSS), pyruvate kinase (PK) andpyrophosphatase (PPA). The flask was placed into an incubator at 16-50°C. with gentle shaking. The reaction was monitored by TLC. More enzymesare added if the reaction stops. The reaction is stopped when no morestarting material is observed by TLC. The SSEA4 product was isolated by¹⁸C reverse phase column in 45% yield.

Allyl-SSEA₄: ¹H NMR (600 MHz, D₂O) δ 6.00 (m, 1H), 5.40-5.37 (d, J=17.3Hz, 1H), 5.30-5.28 (d, J=10.4 Hz, 1H), 4.92 (d, J=3.9 Hz, 1H), 4.70 (d,J=8.5 Hz, 1H), 4.54-4.51 (m, 3H), 4.40-4.38 (m, 2H), 4.25-4.18 (m, 3H),4.10-3.52 (m, 34H), 3.35-3.32 (t, J=8.6 Hz, 1H), 2.77 (dd, J=12.5, 4.6Hz, 1H), 2.03 (s, 6H), 1.80 (t, J=12.1 Hz, 1H); ¹³C NMR (150 MHz, D2O) δ175.2, 175.1, 174.1, 133.4, 121.6, 118.9, 104.7, 103.4, 103.1, 101.1,100.5, 99.8, 79.9, 78.9, 78.8, 77.3, 75.7, 75.5, 75.0, 74.7, 74.6, 73.0,72.9, 72.2, 72.1, 71.9, 71.0, 70.8, 70.4, 69.1, 69.0, 68.5, 68.2, 68.0,67.7, 67.5, 62.6, 61.1, 60.5, 60.4, 60.1, 51.7, 51.4, 39.8, 22.4, 22.1;HRMS (ESI-TOF, M−H) C₄₆H₇₅N₂O₃₄O⁻ calcd for 1199.4196. found 1199.4208.

¹H NMR (600 MHz, D₂O) δ 4.94 (d, J=3.8 Hz, 1H), 4.72 (d, J=8.5 Hz, 1H),4.54-4.50 (m, 3H), 4.40 (t, J=6.4 Hz, 1H), 4.27 (d, J=2.0 Hz, 1H), 4.20(d, J=2.8 Hz, 1H), 4.10-3.54 (m, 37H), 3.34-3.31 (m, 1H), 3.02 (t, J=7.6Hz, 2H), 2.78 (dd, J=12.4, 4.6 Hz, 1H), 2.05 (m, 6H), 1.80 (t, 12.2 Hz,1H), 1.74-1.67 (m, 4H), 1.51-1.45 (m, 2H); ¹³C NMR (150 MHz, D₂O) δ175.0, 174.9, 173.9, 104.5, 103.2, 102.9, 101.9, 100.3, 99.6, 79.7,78.8, 78.7, 77.1, 75.5, 75.4, 74.8, 74.7, 74.6, 74.5, 72.9, 72.7, 72.1,71.8, 70.8, 70.2, 70.0, 68.9, 68.9, 68.3, 68.0, 67.8, 67.5, 67.3, 62.4,60.9, 60.3, 60.3, 60.0, 51.6, 51.3, 39.7, 39.3, 28.1, 26.5, 22.3, 22.0,22.0; HRMS (ESI-TOF, MNa⁺) calcd for C₄₈H₈₃N₃O₃₄Na 1268.4756. found1268.4760.

TABLE 6 Basic composition of glycosphingolipids Globoseries Gal GlcGalNAc GlcNAc Neu5Ac Fuc Globotetraose 2 1 1 0 0 0 (Gb4) Globopentaose 31 1 0 0 0 (Gb5) Globo H 3 1 1 0 0 1 (Fucosyl-Gb5) SSEA4 3 1 1 0 1 0(Sialyl-Gb5) Isoglobotetraose 2 1 1 0 0 0 Neolactoseries 2 1 0 1 1 0Lactoseries 2 1 0 1 1 0 Ganglioseries 2 1 1 0 2 0

TABLE 7 Yields of Each step of glycosylation with regeneration Enzymeinvolvement Product Yield Step 1. GalK, AtUSP, PykF, PPA, LgtC*allyl-Gb3 99% Step 2. NahK, GlmU, PykF, PPA, LgtD* allyl-Gb4 96% Step 3.GalK, AtUSP, PykF, PPA, LgtD* allyl-Gb5  95%** Step 4a. FKP, PykF, PPA,FutC* allyl- 94% Globo H Step 4b. CSS, CMK, PykF, PPA, JT-FAJ-16*allyl-SSEA4 45% *DNA sequences were optimized for E. coli expression.**When using pure allyl-Gb4 as an acceptor.

Example 2 One-Step Synthesis of Allyl-Gb5(SSEA3) from Allyl-Lactose

Allyl-Gb5 was synthesized from allyl-lac via a one-step chain reactionas illustrated in FIG. 6, without purifying any of the intermediates.

5 mmol Allyl-lac, 5 mmol galactose, 12 mmol PEP, 0.25 mmol ATP, 0.25mmol UTP with 10 mM MgCl₂ in 100 mM Tris-HCl buffer (pH 7.5) were mixedin a flask. Enzymatic reaction was initiated by adding into the flask asuitable α1,4-galactosyltransferase (LgtC), GalK, AtUSP, PK and PPA tosynthesize allyl-Gb3. The flask containing the reaction mixture wasplaced in a 16˜50° C. incubator with gently shaking. TLC analysis wasperformed to monitor the synthesis process. If no further synthesis ofallyl-Gb3 is observed, additional enzymes were added.

After synthesis of allyl-Gb3, another set of components, including 5mmol of GalNAc, 12 mmol PEP, and a suitable amount of N-acetylhexosamine1-kinase (NahK-CP), N-acetylglucosamine 1-phosphate uridylyltransferase(GlmU), PK, PPA and β 1,3-N-acetylgalactosaminyltransferase (LgtD), wasadded into the flask. The reaction mixture thus formed was incubatedunder the same conditions under which allyl-Gb3 was synthesis. If nofurther synthesis of allyl-Gb4 is observed, additional amounts of theenzymes can be added.

After synthesis of allyl-Gb4, 5 mmol galactose and 12 mmol PEP wereadded into the flask without purifying the allyl-Gb4. The nextgalactosylation reaction was initiated by adding suitableβ1,3-galactosyltransferase (LgtD), GalK, AtUSP, PK and PPA to synthesizeallyl-Gb5. The flask containing the reaction mixture was placed in a16-50° C. incubator with gently shaking. TLC was performed to monitorthe synthesis process. Additional amounts of enzymes can be added if nofurther synthesis of allyl-Gb5 is observed. The yield of this one-stepsynthesis of allyl-Gb5 from allyl-lac is about 40%.

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Other Embodiments

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

Furthermore, the invention encompasses all variations, combinations, andpermutations in which one or more limitations, elements, clauses, anddescriptive terms from one or more of the listed claims is introducedinto another claim. For example, any claim that is dependent on anotherclaim can be modified to include one or more limitations found in anyother claim that is dependent on the same base claim. Where elements arepresented as lists, e.g., in Markush group format, each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should it be understood that, in general, where the invention,or aspects of the invention, is/are referred to as comprising particularelements and/or features, certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements and/or features. For purposes of simplicity, those embodimentshave not been specifically set forth in haec verba herein. It is alsonoted that the terms “comprising” and “containing” are intended to beopen and permits the inclusion of additional elements or steps. Whereranges are given, endpoints are included. Furthermore, unless otherwiseindicated or otherwise evident from the context and understanding of oneof ordinary skill in the art, values that are expressed as ranges canassume any specific value or sub-range within the stated ranges indifferent embodiments of the invention, to the tenth of the unit of thelower limit of the range, unless the context clearly dictates otherwise.

This application refers to various issued patents, published patentapplications, journal articles, and other publications, all of which areincorporated herein by reference. If there is a conflict between any ofthe incorporated references and the instant specification, thespecification shall control. In addition, any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Because such embodimentsare deemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the invention can be excluded from any claim,for any reason, whether or not related to the existence of prior art.

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments described herein. The scope of the present embodimentsdescribed herein is not intended to be limited to the above Description,but rather is as set forth in the appended claims. Those of ordinaryskill in the art will appreciate that various changes and modificationsto this description may be made without departing from the spirit orscope of the present invention, as defined in the following claims.

1-34. (canceled)
 35. A method for enzymatically synthesizing anoligosaccharide, comprising: (i) producing UDP-GalNAc from GalNAc in thepresence of a set of UDP-GalNAc regeneration enzymes, wherein the set ofUDP-GalNAc regeneration enzymes comprises an N-acetylhexosamine1-kinase, an N-acetylglucosamine 1-phosphate uridyltransferase, apyruvate kinase, and optionally, a pyrophosphatase, and (ii) convertingGb3-OR^(1A) into Gb4-OR^(1A) in the presence of the UDP-GalNAc and abeta-1,3-N-acetylgalactosaminyltransferase, wherein R^(1A) is hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, or an oxygen protecting group.
 36. The method of claim 35,wherein (i) and (ii) occur in a Gb4-synthesis reaction mixturecomprising GalNAc, PEP, ATP, UTP, the Gb3-OR^(1A), thebeta-1,3-N-acetylgalactosaminyltransferase, and the set of UDP-GalNAcregeneration enzymes.
 37. The method of claim 35, wherein thebeta-1,3-N-acetylgalactosaminyltransferase is LgtD from H. influenza,the N-acetylhexosamine 1-kinase is from B. longum, theN-acetylglucosamine 1-phosphate uridyltransferase is from E. coli, thepyruvate kinase is from E. coli, or the pyrophosphatase is from E. coli.38. The method of claim 35, wherein the R^(1A) is hydrogen, allyl,substituted alkyl, biotin, or a ceramide.
 39. The method of claim 35,further comprising isolating the Gb4-OR^(1A).
 40. The method of claim35, further comprising: (iii) converting the Gb4-OR^(1A) intoGb5-OR^(1A) in the presence of UDP-Gal and abeta-1,3-galactosyltransferase.
 41. The method of claim 40, furthercomprising: (iv) producing the UDP-Gal from galactose in the presence ofa set of UDP-Gal regeneration enzymes, wherein the set of UDP-Galregeneration enzymes comprises a galactokinase, an UDPpyrophosphorylase, a pyruvate kinase, and optionally, a pyrophosphatase.42. The method of claim 41, wherein (iii) and (iv) occur in aGb5-synthesis reaction mixture comprising galactose, PEP, ATP, UTP, theGb4-OR^(1A), the beta-1,3-galactosyltransferase, and the set of UDP-Galregeneration enzymes.
 43. The method of claim 40, wherein thebeta-1,3-galactosyltransferase is LgtD from H. influenza; thegalactokinase is from E. coli, the UDP-sugar pyrophosphorylase is fromA. thaliana, the pyruvate kinase is from E. coli, or the pyrophosphataseis from E. coli.
 44. The method of claim 40, further comprisingisolating the Gb5-OR^(1A).
 45. The method of claim 40, furthercomprising: (v) converting the Gb5-OR^(1A) into Fucosyl-Gb5-OR^(1A) inthe presence of GDPFuc and an alpha-1,2-fucosyltransferase.
 46. Themethod of claim 45, further comprising: (vi) producing the GDP-Fuc fromfucose in the presence of a set of GDP-Fuc regeneration enzymes, whereinthe set of GDP-Fuc regeneration enzymes comprises anL-fucokinase/GDP-fucose pyrophosphorylase, a pyruvate kinase, andoptionally, a pyrophosphatase.
 47. The method of claim 46, wherein (v)and (vi) occur in a Fucosyl-Gb5-synthesis reaction mixture comprisingfucose, ATP, GTP, PEP, the Gb5-OR^(1A), thealpha-1,2-fucosyltransferase, and the set of GDP-Fuc regenerationenzymes.
 48. The method of claim 47, wherein the Fucosyl-Gb5-synthesisreaction mixture is prepared by mixing the Gb5-synthesis reactionmixture with at least fucose, GTP, the alpha-1,2-fucosyltransferase, andthe L-fucokinase/GDP-fucose pyrophosphorylase.
 49. The method of claim45, wherein the L-fucokinase/GDP-fucose pyrophosphorylase is from B.fragilis, or the alpha-1,2-fucosyltransferase is from H. pylori.
 50. Themethod of claim 45, further comprising isolating theFucosyl-Gb5-OR^(1A).
 51. The method of claim 40, further comprising:(vii) converting the Gb5-OR^(1A) into Sialyl-Gb5-OR^(1A) in the presenceof CMP-Neu5Ac and an alpha-2,3-sialyltransferase.
 52. The method ofclaim 51, further comprising: (viii) producing the CMP-Neu5Ac fromNeu5Ac in the presence of a set of CMP-Neu5Ac regeneration enzymes,wherein the set of CMP-Neu5Ac regeneration enzymes comprises a cytidinemonophosphate kinase, a CMP-sialic acid synthetase, a pyruvate kinase,and optionally, a pyrophosphatase.
 53. The method of claim 52, wherein(vii) and (viii) occur in a Sialyl-Gb5-synthesis reaction mixturecomprising Neu5Ac, CTP, PEP, the Gb5-OR^(1A), thealpha-2,3-sialyltransferase, and the set of CMP-Neu5Ac regenerationenzymes.
 54. The method of claim 53, wherein the Sialyl-Gb5-synthesisreaction mixture is prepared by mixing the Gb5-synthesis reactionmixture with at least Neu5Ac, CTP, the alpha-2,3-sialyltransferase, thecytidine monophosphate kinase, and the CMP-sialic acid synthetase. 55.The method of claim 51, wherein the alpha-2,3-sialyltransferase is fromM. bacteria, the cytidine monophosphate kinase is from E. coli, or theCMP-sialic acid synthetase is from P. Multocida.
 56. The method of claim51, further comprising isolating the Sialyl-Gb5-OR^(1A). 57-99.(canceled)